Biosystems Solutions - appliedbiosystems.com · Welcome to Biosystems Solutions Today, traditional...

60
Biosystems The Magazine of Integrated Science Solutions Genome-wide Expression Profiling Genome-wide Expression Profiling Announcing the Family of Real-Time PCR Platforms from Applied Biosystems Announcing the Family of Real-Time PCR Platforms from Applied Biosystems Determination of the Doping Agent Tetrahydrogestrinone by LC-APCI-MS/MS Determination of the Doping Agent Tetrahydrogestrinone by LC-APCI-MS/MS in Search of a More Tolerant Immune System FRIEND&FOE: in Search of a More Tolerant Immune System FRIEND & FOE : European Edition Issue 10 Summer 2004

Transcript of Biosystems Solutions - appliedbiosystems.com · Welcome to Biosystems Solutions Today, traditional...

Biosystems

The Magazine of Integrated Science

Solutions

Genome-wideExpression ProfilingGenome-wideExpression Profiling

Announcingthe Family ofReal-TimePCR Platformsfrom Applied Biosystems

Announcingthe Family ofReal-TimePCR Platformsfrom Applied Biosystems

Determination of the Doping AgentTetrahydrogestrinone

by LC-APCI-MS/MS

Determination of the Doping AgentTetrahydrogestrinone

by LC-APCI-MS/MS

in Search of a More Tolerant Immune SystemFRIEND&FOE:in Search of a More Tolerant Immune SystemFRIEND&FOE:

European Edition Issue 10 Summer 2004

applicationsSuccessful connectionsEfficient primary structure elucidation of disulfide-bridged peptides.....................................................................................................................

Determination of the doping agent tetrahydrogestrinone by LC-APCI-MS/MSThe designer steroid tetrahydrogestrinone (THG) was identified in urine samples of elite athletes.......................................................................

Characterisation of the proteome of synaptic proteins from rat brain using cICAT® Reagent-LC MS/MSIn combination with 2D-gel electrophoresis MS and MS/MS...............................................................................................................................

Peptidomics®

Differential analysis and inventory of human cerebrospinal fluid..........................................................................................................................

Determination of residues of chloramphenicol in milk and milk products using HPLC/MS/MSIn the European Union the use of chloramphenicol (CAP) as a veterinary drug for food producing animals is prohibited...................................

Applied Biosystems technology arms researchers in the battle against SARSIn February 2003, a mysterious illness resembling pneumonia began to spread rapidly through different parts of Asia.........................................

High-throughput gene expression systemFor a liver carcinoma research project at CEPH, Paris...........................................................................................................................................

Proteomics Solution in a leading edge research environmentScientists involved in protein expression analysis today have more and more to deal with.....................................................................................

Detecting a peptide biomarker for hypertension in plasmaPeptide quantitation using the 4000 Q TRAP™ System.........................................................................................................................................

Contents

Molecular BiologyProteomics LC/MSInformatics

cover story05FRIEND & FOE:in Search of a More Tolerant Immune SystemImagine being able to modify the immune system to deter its troublesome responses, while leaving its disease-fighting capabilities intact. That’s what the Immune Tolerance Network (ITN), an international network of basic and clinical researchers that specialise in a wide-range of immune-related diseases, hopes to accomplish.

Welcome to Biosystems SolutionsToday, traditional laboratory research is being complemented with information-based science that will open up new horizons. This will have a huge impact on our understanding of biology, medicine and information science.

We are pleased to release the latest issue of Biosystems Solutions, which includes a wealth of knowledge in an exciting and varied mix of articles that reflect scientific research today. This will undoubtedly be a valuable source of information for your life science discovery.

We are sure you will enjoy Biosystems Solutions Issue 10, and as always your comments and suggestions are more than welcome. Contact us at: [email protected]

Cover GraphicCells in the bloodstream with foreign bodies. Red blood cells (red) have a biconcave shape. Neutrophil white blood cells (blue, transparent) have athree-lobed nucleus (pink). These cells are part of the immune response, and help to keep the blood free of foreign bodies (spiky). These representallergens, hormones or viruses.

KeyThe icons below help you to easily see which articles contain information related to Proteomics, Informatics, LC/MS and Molecular Biology.

081012141618212224

02BiosystemsSolutions

Biosystems Solutions

The Magazine of Integrated Science

promotionsSpecial Offers for GeneAmp® PCR Systems and AmpliTaq Gold® DNA PolymeraseDiscounts available until 31 July 2004..........................................................................................................................................................................

Achieve better, faster synthesis and superior peptidesTake advantage of our Special Offer and try out the most versatile activator chemistry, HATU....................................................................................

4446

customer focusGerman researcher wins Applied Biosystems award for excellence in mass spectrometryIn recognition of outstanding work in the area of protein analysis................................................................................................................................

BioQualificationSM ServiceSupporting the Qualification process in your laboratory with the appropriate documentation and agreements.............................................................

High throughput genotyping using ABI PRISM® 7900HT Sequence Detection SystemA partnership between a CNRS laboratory and the Applied Biosystems and Genoscreen companies............................................................................

Genomics SpotlightDr Raija L.P. Lindberg, Head of the Clinical Neuroimmunology Laboratory, University Hospital of Basel..................................................................

47485052

BiosystemsSolutions03

bio-highlightsResearch using the 8500 Affinity Chip AnalyzerPresented at two forthcoming conferences.....................................................................................................................................................................

Applied Biosystems asks >>> What are your Real-time PCR needs?AB-ONLINE >>> News on demand for integrated science............................................................................................................................................

Narrowing in on your mutationsApplied Biosystems now offers two new tools to significantly ease the work and to shorten the time between hypothesis and result............................

LCMS food and environmental applications binderApplied Biosystems Mass Spectrometry systems are helping scientists solve analytical problems....................................................................................

Customer Training CoursesThe knowledge required to achieve the very best results from your research..................................................................................................................

5455565859

applications…continued

Genome-wide expression profiling with the Applied Biosystems Expression Array SystemExpression profiling of over 30,000 genes in paired normal and breast tumour tissue...................................................................................................

Assessment of apoptosis and lead compound cytotoxicityUsing the Applied Biosystems 8200 Cellular Detection System.....................................................................................................................................

2628

product newsNew BioTrekker™ Software v1.0Offers a solution to your genotyping data management problems ................................................................................................................................

NanoMate® 100 SystemFor the QSTAR®, API 3000™, API 4000™ & 4000 Q TRAP™ Systems........................................................................................................................

New amine specific iTRAQ™ ReagentsExpand multiplexing and quantitation capabilities for Proteomic researchers................................................................................................................

Applied Biosystems LIMS for the Life SciencesTransforms the way your laboratory operates.................................................................................................................................................................

Announcing the family of Real-Time PCR platforms from Applied BiosystemsInstruments to suit all requirements..............................................................................................................................................................................

SQL*LIMS® v5 SoftwareThe best in LIMS just got better...................................................................................................................................................................................

Introducing Tempus™ Blood RNA TubesStabilise RNA profiles in whole blood for 5 days!.........................................................................................................................................................

31323436384042

SEE MOREGENES

NEW

!

From the leader in real-tim

e

PCR TaqM

an ® products

04BiosystemsSolutions

The new Applied Biosystems Expression Array System—the mostcomplete and most sensitive human genome expression analysissystem available. Thanks to advanced design and novel chemiluminescence

chemistry, now you can detect expression events at lower levels and on more genes than

ever before—using a minimal amount of sample. With one integrated workflow and a

single microarray built on today’s most complete dataset, you get unambiguous, high-quality results that drive increased research

productivity. And to accelerate your decision making even faster, the system comes complete with valuable functional

information and annotation, links to the Celera Discovery System™, plus off-the-shelf TaqMan® Gene Expression Assays matched

to the arrays. Want to see more? For information and free webinars visit: http://info.appliedbiosystems.com/microarray

iScience. Applied Biosystems provides the innovative

products, services, and knowledge resources that are

enabling new, integrated approaches to scientific discovery.

Use less sample.

cover story

BiosystemsSolutions05

magine being able to modify the immune system to

deter its troublesome responses, while leaving

its disease-fighting capabilities intact. That’s what

the Immune Tolerance Network (ITN), an international

network of basic and clinical researchers that

specialise in a wide-range of immune-related diseases,

hopes to accomplish.

ITN has teamed with Applied Biosystems in a multi-year

research agreement to help elucidate the molecular

mechanisms underlying immune tolerance and provide a

basis for developing methods to induce such tolerance.

ITN works with Applied Biosystems instrument

technologies, biological content and services, operating in

concert to provide an Integrated Science (iScience)

solution for researchers studying gene expression.

I

Friend & Foe:in Search of a More Tolerant Immune System

Page 6

Mark Springer, BioBeat® Online Magazine(www.biobeat.com)

A world-class contract research group equipped

with advanced high-throughput analysis platforms,

high-quality biological content, and industry-leading bioinformatics

Superior alternative to immunosuppressive agents

The challenge for the ITN scientists is to develop, implement,

and assess strategies that enable the immune system to

become tolerant to specific antigens. “What we’re trying

to achieve is a therapeutic that will allow people to

eliminate the use of lifelong immunosuppressants,”

says Dr. Vicki Seyfert-Margolis, the ITN Executive Director

of Tolerance Assays. “That may mean you take a short-term

therapy to induce immune tolerance, and then you’re drug-free

for the rest of your life.” If successful, it would transform

the practice of organ transplants and provide a superior

alternative to immunosuppressive agents in the treatment of

autoimmune diseases.

“The clinical investigators see the care that the ITN has taken

in selecting Applied Biosystems. They have a high comfort

level and excitement about sending their specimens to them for

analysis.” says Dr. Seyfert-Margolis.

Genetic markers for immune tolerance

The first step of the project was to hunt for genetic markers for

immune tolerance. The ITN and Applied Biosystems'

scientists relied extensively on the Celera Discovery System™

(CDS) Online Platform. Starting with a list of a few hundred

genes from the ITN, the scientists performed homology

searches of CDS content to generate a comprehensive list of

possible immune tolerance genes by selecting members within

gene families. CDS consolidates high-quality biological content

with visualisation and analysis tools on one web site to make the

job easier. The CDS PANTHER™ Protein Classification

System, for example, categorised the genes’ protein products by

biological process and molecular function. The original list of

genes expanded to 1,000 related genes thought likely to be

involved in the modulation of the immune response.

Finding genetic markers for tolerance

Finding genetic markers for immune tolerance will help

researchers expand the range of therapeutic options available

to clinicians for treating a host of autoimmune diseases,

and different kinds of allergies, as well as for helping to prevent

rejection of transplanted organs. However, the quest to find

such helpful markers has just begun.

"There are not yet any genetic markers for immune tolerance.

If we could find markers that were in fact indicative of

clinical tolerance, it might lead us to a clearer quantitative

definition of tolerance," says Dr. Seyfert-Margolis.

According to Dr. Seyfert-Margolis, a good genetic marker is

one that makes it possible to determine if someone is clinically

tolerant of a foreign antigen. Tolerance may be indicated by a

shift in the gene expression profiles of cytokines, or other genes

may be involved.

"If those genes are genes that we know a lot about

immunologically, we may be able to understand how it

[tolerance] works," she says.

Even if some of the 1,000 genes turn out to not have a known

role in the immune system, they may still be valuable markers,

because their patterns of expression may be correlated with

different aspects of a disease.

With regard to analysis of gene expression patterns in clinical

specimens, according to Dr. Seyfert-Margolis, the ITN is using

both real time RT-PCR assays and microarray analysis in

parallel on the same clinical specimens.

"We hope to find [gene expression] patterns that correlate

with clinical changes," says Dr. Seyfert-Margolis. "If we're just

asking if there is going to be an increase in particular cytokines,

those questions we can answer very soon. Whether or not

that means you've achieved tolerance or not I don't know.

All of our studies are two or three years in duration."

One of the biggest challenges for the ITN, but also perhaps one

of the most alluring possible outcomes of these gene expression

studies will be an interpretation of the collected data that leads

to a better understanding of how immune tolerance works.

"Finding the mechanism of action will be difficult.

It will require sharing of information and then having

investigators go back to the bench," notes Dr. Seyfert-Margolis.

Using genetic markers for tolerance

The goals of the ITN, however, go beyond understanding

how tolerance works. They extend to modifying the ways in

which the immune system responds to an antigen. Already,

the ITN is conducting studies of the effects of immune

tolerance in organ transplants, allergies, and autoimmune

diseases such as type 1 diabetes and multiple sclerosis (MS).

For example, for organ transplants, the ITN plans to use

real-time RT-PCR assays to look at a number of genes that it

believes are involved in the prediction of acute rejection events.

"There are a couple of clinical trials coming up in which

clinical investigators will be performing organ transplants in

people who will be given an experimental therapeutic and a

single immunosuppressive agent," notes Dr. Seyfert-Margolis.

"The clinicians will withdraw the immunosuppressive agent

and see if the patients are tolerant. And, in that context,

it's going to be very important to know any piece of

information that would be predictive of an acute rejection

event, before we see pathology."

Any discoveries about immune tolerance in the transplant

studies could perhaps also benefit people afflicted with

autoimmune diseases such as MS, and type 1 diabetes.

06BiosystemsSolutions

cover story

If successful, it would transform the practice of organ transplants

and provide a superior alternative to immunosuppressive

agents in the treatment of autoimmune diseases

a good genetic marker is one that makes it possible to determine

if someone is clinically tolerant of a foreign antigen

“Autoimmune diseases are multifactorial and multiple genes are involved.

So, these types of diseases are going to require multiple approaches,

multiple assays, and probably a multivariant sort of analysis”

INFO

For more information on:ABI PRISM® 7900HTSequence DetectionSystem enter: 101

For more information on:ABI PRISM™ 6100Nucleic Acid PrepStationenter: 102

For more information on:Tempus™ Blood RNAPurification Tubes enter: 103

Related articles can be found on:Pages 38 & 42Contact

usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]

BiosystemsSolutions07

Although fairly common, autoimmune diseases are difficult to

define precisely.

"With autoimmunity, you don't know where you are

at the stage of the disease and when people are presenting,"

Dr. Seyfert-Margolis notes. "Autoimmune diseases are

multifactorial and multiple genes are involved. So, these types

of diseases are going to require multiple approaches, multiple

assays, and probably a multivariant sort of analysis."

"I think that the genetics in all of these autoimmune diseases,

such as large studies to look for SNPs (single nucleotide

polymorphisms), are going to be very difficult," she adds.

"Finding molecular markers may help to clarify the situation,

because, to date, there just aren't any good immunological

markers for most of these diseases."

Integrating products and services

Over 22,500 pre-designed, validated, off-the-shelf gene

expression assays are currently available online, with plans

to provide assays for every gene in the human genome

(www.allgenes.com). Approximately two-thirds of the

ITN assays are available as TaqMan Gene Expression Assays

through the Applied Biosystems e-commerce web site

(http://myscience.appliedbiosystems.com). The Custom

TaqMan Assays from Applied Biosystems provided the balance

with custom-made gene expression assays.

For sample preparation, the ITN chose labs that use the

new Applied Biosystems Tempus™ Blood RNA Purification

Tubes for mRNA purification and the ABI PRISM™ 6100

Nucleic Acid PrepStation for sample preparation. The sample-

prep labs send the samples to a service laboratory, which in

turn performs quantitative gene expression assays using the

ABI PRISM® 7900HT Sequence Detection System.

The ITN/Applied Biosystems team opted to perform the assays

using the TaqMan Low Density Array format rather than in

tube format. Each of the 384 bubble-wells of the card contains

the primer and probe sets for the gene expression assays.

The service laboratory just adds samples and TaqMan®

Universal PCR Master Mix to the eight channels of the card,

performs a brief centrifugation step, seals the array and

analyses it on the 7900HT real-time PCR system.

Improving therapeutic options

The ITN intends to use the gene expression data derived from

these studies to assess relative expression levels of genes

between normal controls and patients, and in individual

patients at various time points throughout the clinical trials.

The data are expected to track parameters such as disease onset,

disease progression, and patient responses to therapeutics.

Ultimately, this marriage of clinical and genetic sciences

applied to immune tolerance is designed to improve

therapeutic options in organ transplantation, autoimmune

diseases, allergy and asthma.

For further information go to:http://europe.appliedbiosystems.com/biobeat/itn

Applied Biosystems Technology:

Informatics

» Celera Discovery System™ Online Platform

» PANTHER™ Protein Classification System

» SQL*LIMS® Software

Real-Time PCR

» TaqMan® Gene Expression Assays

» Custom TaqMan® Assays

» ABI PRISM® 7900HT Sequence Detection System

» TaqMan® Low Density Array

Sample Preparation

» ABI PRISM™ 6100 Nucleic Acid PrepStation

» Tempus™ Blood RNA Purification Tubes

Right.

Dr. Vicki Seyfert-Margolis ITN Executive Director

of Tolerance Assays

omplete de novo primary structure elucidation of highly disulfide-bridged neurotoxic peptides from the spider Cupiennius salei is demonstrated,

using a combination of Edman sequencing on the PROCISEcLC 492 sequencing system and tandem mass spectrometryon the QSTAR Pulsar hybrid quadrupole time-of-flight mass spectrometer. The use of these complementarytechnologies provides unambiguous analytical data for successful characterisation of peptides and proteins, while simultaneously extending the range of applications.

Introduction

N-terminal amino acid sequence analysis by Edmandegradation has been the working-horse for the de novosequence determination of peptides and proteins for manyyears. Modern sequencing systems based on the Edmanchemistry provide high sensitivity and enable fully automatedanalyses. Due to the unambiguous sequence data generated,Edman sequencing is still the reference technology forcomplete de novo protein sequencing. As a further keytechnology, mass spectrometry has entered the field ofbioanalysis within the last decade, enabling rapid proteinidentification. The impressive resolving power and massaccuracy of modern mass spectrometric instrumentation offers great potential for elucidation of structural details. The combination of Edman sequencing and tandem massspectrometry is an efficient approach to the primary structureelucidation of complex, highly disulfide-bridged spider toxins.

Methods

Edman sequencing: The sequences of two reduced andalkylated neurotoxic peptides CSTX-1 (8,351.90 Da) andCSTX-9 (7,530.25 Da) were determined by Edmandegradation on a Procise cLC 492 protein sequencer1,2,3. The overlaps were secured by a combination of enzymaticcleavages with Asp-N, chymotrypsin, and immobilised trypsin (figure 1). The disulfide-containing peptides weregenerated by cleaving native CXTX-1 and CSTX-9 withimmobilised trypsin.

Electrospray tandem mass spectrometry: Identification of thedisulfide-bridge pattern of the peptides was performed bynano-electrospray tandem mass spectrometry on the QSTARPulsar hybrid quadrupole time-of-flight mass spectrometer2,4.The purified tryptic peptides, consisting of four to five chainscross-linked by the original disulfide bridges, were subjected toMS and MS/MS analysis.

Results and Discussion

The complete sequences of CSTX-1 and CSTX-9 with thecorresponding overlaps are shown in figure 1. The proximity ofthe cysteine residues and the absence of suitable cleavage siteswithin the disulfide-containing sequences caused the classicalapproaches for elucidation of the disulfide-bridge pattern to fail.

08BiosystemsSolutions

INFO

For more information on:Efficient primarystructure elucidation of disulfide-bridgedpeptides enter: 104

appl icat ions

C

SuccessfulConnections

Efficient primary structure elucidation of disulfide-bridged peptides using the PROCISE® cLC sequencing system and the QSTAR® Pulsar system

Stefan Schürch1, Johann Schaller1, Urs Kämpfer1, Lucia Kuhn-Nentwig1, Stefan König2

1. University of Bern, Bern, Switzerland 2. Applied Biosystems, Rotkreuz, Switzerland

BiosystemsSolutions09

Consequently, the basically unspecific gas-phase dissociation ofthe disulfide-linked peptides by tandem mass spectrometry isan attractive alternative to the classical procedures.Identification of the disulfide bridge pattern was based onobservation of characteristic fragment ions generated bydissociation of the peptide bonds adjacent to the cysteines, asillustrated for CSTX-9 in figure 2. The identification of thedisulfide-bridge pattern of CSTX peptides by tandem massspectrometry represents a valuable alternative to traditionalmethods for elucidation of complex molecular structures.Modern high-performance instrumentation, such as theQSTAR Pulsar mass spectrometer, greatly enhance thepotential of this technique, since a wide range of informationis obtained from a single experiment using minute amounts ofsample only. Sample preparation is essentially reduced to apurification step, thus, avoiding the risk of potential structuralalteration, e.g. disulfide scrambling.

Conclusions

The results demonstrate that Edman sequencing and tandem mass spectrometry are by no means competingtechniques. These two techniques can be considered ascomplementary tools, each one offering its particularadvantages. Edman sequencing is a straightforward andefficient approach to the determination of amino acidsequences of peptides and proteins, whereas tandem massspectrometry exhibits its ultimate potential for the elucidation of structural details. If both techniques are available to the researcher, bioanalytical problems are solved most accurately and efficiently.

References:

1. L. Kuhn-Nentwig, J. Schaller, W. Nentwig, Toxicon, 1994, 32, 287-302.

2. J. Schaller, U. Kämpfer, S. Schürch, L. Kuhn-Nentwig, S. Haeberli, W. Nentwig, Cell Mol. Life Sci., 2001, 58, 1538-1545.

3. J. Schaller, L. Kuhn-Nentwig, S. Schürch, U. Kämpfer, J. Müller, W. Nentwig, Chimia 2001, 55, 1058-1062.

4. S. Schürch, J. Schaller, U. Kämpfer, L. Kuhn-Nentwig, W. Nentwig,Chimia 2001, 55, 1063-1066.

Figure 2.

Product ion spectra are characterised by abundant y-type ions due to cleavage of theunbridged portions of the peptide chains. Bridge-defining fragment ions of higher chargestate are identified unambiguously due to the high resolving power and mass accuracy of the QSTAR® Pulsar mass spectrometer. The disulfide-bridge pattern corresponds to the inhibitor cystine knot (ICK) structural motif.

Figure 1.

Amino acid sequences of CSTX-1 (top) and CSTX-9 (bottom) determined by Edmandegradation. Positions sequenced are indicated by horizontal bars. Up to 53 amino acidswere sequenced in a single run.

Contact

usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]

10BiosystemsSolutions

Determinationof the Doping Agent

Tetrahydrogestrinoneby LC-APCI-MS/MS

Introduction

Gestrinone (figure 1, 1) is a drug administered in cases of endometriosis1. Its hydrogenation at the ethinyl residue at carbon 17 results in a steroid hormone termedtetrahydrogestrinone (THG), the physiological effects of which have not been investigated until now. The identificationof the designer steroid THG (figure 1, 2) by researchers of the doping control laboratory of the University of California, Los Angeles (UCLA), has triggered an avalanche in sport as well as the scientific world2-3. The hydrogenation of gestrinonegives rise to a compound related to anabolic steroids, and theshift of four amu compared to gestrinone invested it withinvisibility for conventional multiple reaction monitoring(MRM) screening procedures. With the knowledge of structureand molecular weight of THG and its synthesis as referencecompound, an identification in human urine has becomefeasible by LC-APCI-MS/MS, and its determination isincluded in established procedures covering anabolic steroidssuch as trenbolone and structural analogues.

T

appl icat ions

For more information on:API 2000 LC/MS/MSSystem enter: 105

Related article can be found on:Page 16

AcknowledgementsMario Thevis andWilhelm Schänzer,Institute ofBiochemistry, GermanSport University, Cologne, Germany

Abstract

he designer steroid tetrahydrogestrinone (THG) was identified in urine samples of elite athletes in the doping control laboratory in the United States of America. Hence, all doping control samples have to be analysed in this regard requiring reliable and robust assays, which have been established employing liquid

chromatography/tandem mass spectrometry. Utilising an API 2000™ LC/MS/MS System, THG was determined atconcentrations of 2 ng per mL of human urine by means of multiple reaction monitoring of characteristic product ions.

INFO

Doping control urine samples are tested for the presence of THG

and other prohibited compounds in a screening procedure

employing two principal sample preparation steps

BiosystemsSolutions11

Figure 1.

Chemical structures of gestrinone (1, mol wt = 308), and tetrahydrogestrinone (2, mol wt = 312).

Figure 2.

ESI-product ion spectrumof (M+H)+ of gestrinone(m/z 309, a) andtetrahydrogestrinone (m/z 313, b) at collisionoffset voltages of 40.

Figure 3.

Extracted ionchromatograms of a) urine sample fortified with 2 ng/mL of THG, and b)blank urine specimen.

Methods

Doping control urine samples are tested for the presence ofTHG and other prohibited compounds in a screeningprocedure employing two principal sample preparation steps.Urine specimens are treated with β-glucuronidase from E. coliin order to hydrolyse glucuronic acid conjugates of THG and itsmetabolites, and analytes of interest are extracted from urinewith n-pentane. The organic layer is separated, evaporated todryness, the residue is reconstituted in methanol, and thesample is analysed by LC-APCI-MS/MS. The analyticalinstrumentation consists of an Agilent (Waldbronn, Germany)1100 Series HPLC equipped with a Merck (Darmstadt,Germany) Purospher Star 18e column (4 x 55 mm, particle size3.5 µm), which is interfaced to an API 2000 triple quadrupolemass spectrometer. The API 2000 system is operated in thepositive MRM mode, and for THG two diagnostic iontransitions (m/z 313 – 241, m/z 313 – 199) are recorded.

Results and discussion

THG was synthesised from gestrinone using an adequatecatalyst and hydrogen. The reference compound was used to optimise instrument parameters such as declusteringpotential, collision energies and exit potentials of the API 2000 system, resulting in sufficiently sensitive screeningand confirmation procedures.

In figure 2, the product ion mass spectra of gestrinone andTHG are shown, both containing the characteristic fragmentions at m/z 241 and 199 after ESI and collisionally activateddissociation. These ions are targets of MRM analyses of dopingcontrol urine samples, and, in combination with retentiontimes, signals reliably indicate the presence or absence of THG.A confirmatory analysis in case of suspicious results is based onat least three diagnostic ion transitions that have to meet thecriteria of the International Olympic Committee (IOC) andWorld Anti-Doping Agency (WADA). Figure 3a demonstratesa typical extracted ion chromatogram of a urine sample fortifiedwith 2 ng of THG per mL of urine, which, in comparison to a blank specimen in figure 3b, contains distinct peaks at 13.5 min at the respective ion transitions.

Since the discovery of THG in October 2003, numeroussamples that have been analysed and reported already, were re-analysed in terms of the presence of THG, and the assay based on LC-APCI-MS/MS has proven to be a robust tool generating reproducible results for the detection andidentification of this particular designer steroid in combinationwith other prohibited drugs.

References

1. Martindale – The Extrapharmacopoeia, 30th edition, James E. F. Reynolds (Ed.), Pharmaceutical Press, London, 1993, p. 1185

2. Knight, J. Nature 425, 752 (23 Oct 2003) News

3. Knight, J. Nature 426, 114 - 115 (13 Nov 2003) News Feature

Contact

usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]

Characterisation of the proteome of synapticproteins from rat brain using cICAT® Reagent-LC MS/MS in combination with 2D-gelelectrophoresis MS and MS/MS

Overview

he brain is the most complex and dynamic of all organs with a high degree of computation capability that enables an animal to perceive and integrate

information and to respond to environmental and physiologicalinputs. Central to the neuronal circuitry of the brain is theextensive synaptic connections via which neurotransmissionoccurs. Synaptic neurotransmission involves the release oftransmitter from the pre-synaptic compartment, and theactivation of transmitter receptors and signal transductioncascades in the post-synaptic density (PSD) of the synapticspine. It is generally accepted that information storage in thebrain is established via structural and molecular changes ofsynapses, which arise at least in part from the alteration of protein constituents in the synapse. As a first step towards the understanding of synaptic function and plasticity, we applied 2-D gel-based and cleavable Isotope Coded AffinityTag (cICAT)-based technologies to characterise the proteome ofthis organelle1.

Approach and Results

Purification of the PSD

We used a two-step detergent extraction in conjunction with asucrose gradient ultra-centrifugation to purify the PSD fraction,which also contains a low level of some pre-synaptic proteins.Forebrains from adult rats were homogenised in a sucrosesolution and centrifuged at low speed to remove the nucleusand the intact cells. The supernatant was centrifuged at a higherspeed to obtain a P2 pellet. Synaptosomes, which contain the pre- and post-synaptic compartments, were isolated fromthis P2 pellet by ultra high-speed centrifugation in a sucrosegradient, and then extracted twice with Triton-X 100. After a final high-speed centrifugation the PSD fraction, which constitutes a compact detergent insoluble proteincomplex, was recovered from the pellet phase.

Separation/fractionation of the PSD

and analysis by mass spectrometry

To identify the proteins of the PSD fraction, two separationapproaches have been followed, namely 2-D gel electrophoresisand cICAT Reagent liquid chromatography, which werefollowed by mass spectrometric analyses.

2-D gel electrophoresis and sample

preparation for mass spectrometry

For 2-D gel electrophoresis separation of proteins, samples were solubilised in lysis buffer for 30 min and then used for therehydration and simultaneous loading of the proteins to theIPG strip, which was then focused for 65000V/hr. The seconddimension separation was run overnight in 11% gels. After electrophoresis, gels were fixed and stained using colloidalCoomassie brilliant blue G-250. All the visible protein spotsfrom the Coomassie blue stained gel were manually excised,destained and dried. The gel pieces were then rehydrated in 8 µltrypsin solution (20 µg/ml). After incubation overnight at roomtemperature, 0.5 µl of the incubation buffer was pipetted to theMALDI plate and mixed with 1 µl α-cyano-4-hydroxycinnamicacid. The samples were analysed with a MALDI TOF/TOFmass spectrometer, the 4700 Proteomics Analyzer withTOF/TOF™ optics from Applied Biosystems. In cases where themass spectrometric signals were weak, the remaining incubationbuffer was loaded into a C18 Ziptip® (Millipore) according to theprotocol provided. The bound peptides were eluted from the Ziptip using 1.0 µl of α-cyano-4-hydroxycinnamic acid,which was directly deposited onto the MALDI plate. The trypsin auto-digestion fragments at the protonated massesof 842.510 Da and 221.104 Da, were used as internal standards for the calibration of the MS spectra. All the massspectra were searched against the NCBI database, using onlineMASCOT® software.

Mass spectrometry identification results

by 2-D gel electrophoresis

The identification of the proteins is based on the MASCOTscore of the peptide mass fingerprint and of the daughter ionspectra of the tryptic peptides. In total 250 protein spots wereanalysed. Interestingly, in about 35% of the cases single proteinsare present in 2 or more spots. As an example synapsin II isdetected in 14 spots, from which two groups can be clusteredtogether. The group of proteins that are smaller and generallymore acidic represents one of the alternative spliced forms ofsynapsin II, named synapsin IIb (figure 1). Within both groups,trains of spots that differ in pH can be discerned. This can beexplained in part by the differential phosphorylation ofmultiple phosphorylation sites, through which a progressivedecrease in isoelectric point of the protein resulted. In thisrespect 14 serine, 4 threonine and 2 tyrosine sites on synapsinIIb are predicted to be the phosphorylation sites.Although functional implications of the dynamic anddifferential regulation of the phosphorylation of synapsins havebeen well documented, due to the complexity of the eventsmany of the phosphorylation sites and their combinatorialexistence on single protein remain to be characterised.

T

12BiosystemsSolutions

The identification of the proteins is based on the MASCOT score

of the peptide mass fingerprint and of the daughter

ion spectra of the tryptic peptides

appl icat ions

INFO

For more information on:Cleavable ICAT enter: 106

Related article can be found on:Page 34

Ka Wan Li1and Martin P. Hornshaw

2

1. Faculty of Earth and Life Science, Free University, Amsterdam, The Netherlands. 2. Applied Biosystems, Warrington, UK

Other posttranslational modifications of PSD proteins have alsobeen reported and may contribute to the appearance of some ofthe multiple spots on the 2-D gel. Table 1 summarises the list of proteins that show multiple spots (>5 spots per protein)on the 2-D gel.

cICAT Reagent derivatisation, separation and

mass spectrometry identification results

by cICAT Reagent/liquid chromatography

2-D gel electrophoresis–based methodology is a widely usedtechnique for proteomics studies. However, in our study somemajor PSD proteins are under-represented, for example,glutamate receptor subunits are not detected although they areknown to be highly enriched in the PSD fraction. To addressthis problem, we performed an alternative experiment thatcombined the cICAT Reagent derivatisation/purificationprotocol of the tryptically digested PSD to nano-liquidchromatography-electrospray tandem mass spectrometry.

Briefly, the purified sample was labelled with the cICATReagent and then digested with trypsin to produce trypticpeptides. The cICAT Reagent generates cysteine-containingpeptides that can be affinity selected from non-derivatisedpeptides using avidin chromatography, substantially reducingsample complexity. After elution from the avidin affinitycolumn, the cysteine-containing peptides were resolved by a C18

column (75 µm id, flow rate 200 nl/min, LC Packings) andsprayed on-line into an electrospray tandem mass spectrometer from Applied Biosystems (QSTAR® Pulsar i LC/MS/MSsystem) that automatically performs information dependenttandem MS acquisitions. The spectra were searched against the Pro ICAT engine. In addition to the cytosolic proteins andcytoskeletal proteins, the cICAT Reagent LC-MS/MSexperiment allows the detection of hydrophobic proteins such as the ionotropic glutamate receptors and ion channels,large proteins (plectin > 500 kDa) and basic proteins (ribosomal protein > pH 10) that were missed by the 2-D gelapproach. On the other hand, the different forms ofposttranslationally modified proteins, as trains of protein spots on the 2-D gel, cannot be detected.

Concluding remarks

2-D gel and cICAT Reagent LC based proteomics approaches are complementary and detect overlapping sets of proteins (figure 2). Together, we show that the PSD contains a highdiversity of functional classes of protein. This is in agreementwith previous studies that showed the possible extension of thePSD into the synaptic spine compartment; the spine apparatus,polyribosomes and specialised endocytic zones are foundlocated in close vicinity to the PSD that together with other“cytosolic” proteins such as glycolytic enzymes and signallingenzymes may be interconnected to the PSD by actin filament.Together, we postulate that the PSD is a complex organelleharbouring diverse physiological functions, which puts the PSD into a central position for the autonomous functioning ofthe synaptic spine.

References 1. Ka wan Li, Martin P. Hornshaw, Roel C. Van der Schors,

Rod Watson, Connie, R. Jimenez, Yvonne Gouwenberg, Eckart D. Gundelfinger, Karl-Heinz Smalla, Stephen Tate, August B. Smit (2004) Proteomic analysis of rat brain postsynaptic density: implications of the diverse protein functional groups observed for the integration of synaptic physiology. J. Biol. Chem. 279, 987-1002.

Figure 1.

Synapsin IIb (indicated by arrows) appeared as multiple spots on the 2-D gel.

Figure 2.

Functional grouping ofPSD proteins identifiedby the 2-D gel based (A)and cICAT Reagent LCbased (B) approaches.

Table 1.

Proteins from the PSDfraction that are detectedon the 2-D gel asmultiple spots.About 35% of the distinctproteins were representedby two or more proteinspots. Here, only thosethat are presented as 6 or more spots on the 2-D gel are shown.

BiosystemsSolutions13

Protein name Number of spots

SAP90/PSD-95 6

Vesl-1L/Homer 1 6

CaM kinase II α 6

Actin β 10

Tubulin α 13

Tubulin β 18

Neurofilament, light 10

Internexin α 11

Glyceraldehyde-3-phosphate dehydrogenase 7

Aldolase 6

Synapsin I 9

Synapsin II 14

Left.

Members of theneuroproteomics group at the faculty of Earth and Life Sciences of theVrije UniversiteitAmsterdam. From left to right: K.W. Li, C.R. Jimenez,A.B. Smit, Y. Gouwenberg, R.C. Van Der Schors, Z. El Filai.

14BiosystemsSolutions

appl icat ions

Peptidomics®

Differential Analysis and Inventoryof Human Cerebrospinal Fluid

Introduction

eptides and small proteins, such as hormones, cytokines and growth factors play an active role in many physiological processes. Moreover, as products

of protein degradation and processing, peptides are part ofproteometabolism. Thus, they can reflect disease-relatedchanges in an organism’s homeostasis in several ways. A peptidesource of high diagnostic value is human cerebrospinal fluid(CSF), in which peptides are mainly derived from the centralnervous system (CNS) or from blood.

Material and Methods

Differential Peptide Display® (DPD)-Analysis Peptides wereextracted from raw samples of CSF (0.5ml each) andfractionated in 96 fractions by reversed-phase chromatography.All fractions were measured by Matrix-Assisted LaserDesorption/Ionization Time-Of-Flight mass spectrometry(MALDI-TOF-MS, Voyager-DE™ STR, Applied Biosystems,Framingham, MA, USA) resulting in a peptide massfingerprint with each peptide precisely characterised by itsrelative molecular mass, elution/retention time duringchromatography and signal intensity. The 96 MALDI-MSspectra from each sample were assembled and visualised as 3-dimensional diagrams similar to “2D-Gel Pictures” (figure 1). This was done by Spectromania®, a purpose-builtsoftware package for data visualisation and sophisticatedstatistical analysis of multiparametric data sets1.

Peptide Identification

For identification purposes sample amounts of 4-20 ml CSF were prepared according to the DPD analysis, which was possible due to our scalable preparative process. Peptide identification was performed by means of offline nanoESI-MS/MS (QSTAR® Pulsar System from Applied Biosystems/MDS SCIEX, Concord, Canada) oronline nanoHPLCESI-MS/MS. Generated MS/MS spectrawere mass deconvoluted, and submitted to the MASCOT®

database engine.

PFigure 1.

Peptide master maps representing the CSF peptidome from healthycontrols (A) and patients with primary CNS lymphoma (PCNSL; B).The comparison of the master maps resulted in a heat map. Shown is a detail with the relative mass range between 1,200 and3,000. The average intensities from both sets were compared for everysingle signal position calculating the absolute difference. Signals withthe same intensity in both sets are represented by white spots, whereasred and blue colour indicate stronger signals from PCNSL patients andcontrols, respectively2.

INFO

For more informationvisit:http://www.biovision-discovery.de/set_all.htm

Related article can be found on:Page 24

Peptides and small proteins, such as hormones,

cytokines and growth factors play an active

role in many physiological processes

Relative molecular mass/charge (750 - 15,000)

elut

ion

time

(fra

ctio

ns 1

-96)

Relative molecular mass/charge (1,200 - 3,000)

elut

ion

time

Relative molecular mass/charge (750 - 15,000)

elut

ion

time

(fra

ctio

ns 1

-96)

AcknowledgementDr Peter Schulz-Knappe, BioVisioN,Germany

BiosystemsSolutions15

Results

DPD-guided identification of peptides in human CSF was carried out to investigate several diseases of the CNS. For example,this approach led to novel biomarker panels in the field of Alzheimer’s Disease (AD3) and oncology (PCNSL2): Among others, the peptide-biomarker panel of AD consist of fragments derived from neurosecretory protein VGF, osteopontin and complement C33. As a major difference, a fragment of serum albumin precursor (ALBU 25-48) (figure 1)was identified in the PCNSL-study. The increase of the albumin fragment resulted from a bloodbrain barrier disruption,typically occurring in these tumour patients.

Peptide Inventory of human CSF

The separation into 96 fractions revealed the presence of morethan 6,000 signals in the mass spectrometric analysis, which are inpart redundant due to multiple charge states, oxidation productsor analytical derivatives like sodium adducts, loss of water etc.Here, the Peptide Inventory was created by analysing every secondfraction using nanoHPLC-MS/MS or offline nanoESI-MS/MS.Table 1 gives an extract of the most abundant protein precursors with regard to the number of identified fragments.

Identified peptides were annotated on a CSF master map, which is shown in figure 2.

In addition to their relevance as diagnostic markers, the peptidesrepresent insights into the proteometabolism in CSF. The observedcleavage sites with regard to the precursors indicate differentenzymatic processing motifs in CNS, CSF or in blood. For example, we observed that several fragments were releasedfrom their precursor by cleavage at mono- and dibasic sites. This is indicative for neuroendocine prohormone processing and in accordance with results found by other groups4.

Summary and Conclusions

» A comprehensive Peptide Inventory of a biological source like CSF the determination of prominent landmarks allows a general understanding of the biological source to be achieved

» Due to our highly reproducible and scalable process it is possible to assign Peptide Inventory data to DPD data and vice versa

» DPD technology can be used for the systematic analysis and visualisation of the peptide content of CSF using small sample amounts

» Peptidomics technologies allow the identification of peptides with intrinsic biological relevance important for diagnosis and/or therapy

References

1. P. Schulz-Knappe, H.-D. Zucht, G. Heine, M. Juergens, R. Hess,

M. Schrader, Comb. Chem. High Throughput Screen. 4 (2001) 207.

2. G. Heine, H.-D. Zucht, M.U. Schuhmann, K. Buerger, M. Juergens,

M. Zumkeller, C.G. Schneekloth, H. Hampel, P. Schulz-Knappe, H. Selle,

J. Chromatogr. B 782 (2002) 353.

3. Patent No. WO 02/082075 A2, WO 02/090974 A2, WO 03/048775 A2.

4. M. Stark, O. Danielsson, W.J. Griffiths, H. Jörnvall, J. Johansson, J. Chromatogr. B 754 (2001) 37.

Figure 2.

Annotated peptide master map of human CSF. Each symbol represents an identified peptide.

Contact

usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]

No. Protein precursor Accession No. No. of identified

fragments

1. Neurosecretory protein VGF precursor 015240 35

2. Serum Albumin precursor P02768 22

3. Secretogranin I precursor P05060 20

4. Fibrinogen alpha/alpha E-Chain precursor P02671 17

5. Transthyretin precursor P02766 15

6. Osteopontin precursor P10451 11

Table 1.

Abstract

n the European Union the use of chloramphenicol (CAP) as a veterinary drug for food producing animals is prohibited. For that reason a rapid and sensitive method

for the identification and quantification of CAP in milk andmilk products using LC/MS/MS has been developed.

Blank milk samples were spiked with CAP in theconcentration range of 0.05 - 1.5µg/L. The clean up is based on extraction with acetonitrile, followed by defattingwith chloroform and Solid Phase Extraction on a styrene-divinyl-benzene polymer cartridge. The final extractwas analysed by LC/MS/MS using negative Electrosprayionisation. The method was validated according to the new EU regulation (657/2002/EC).

Introduction

Chloramphenicol (CAP), an antibiotic was formerly usedwidely in veterinary medicine, due to its broad range of activityand low cost. The treatment of animals used for production offood with CAP is prohibited in the European Union andUnited States because of the side effects in humans, in particular the dose independent fatal aplastic anaemia.

Thus, it is necessary to control residues of CAP in edibletissues, like milk and milk products.

Methods to detect CAP in biological matrices especially inmilk described in the literature include immunoassays1,GC/MS2-4 and LC5-6. The immunological methods are suitable for screening purposes, whereas mass spectrometricmethods are used for confirmation analysis. LC/MS can be used without derivatisation, while silylation is necessary for GC/MS.

Therefore, a rapid and sensitive method for the identificationand quantification of CAP in milk and milk products using LC/MS/MS has been developed. The advantage of the described method is its high recovery and the short time of analysis.

Experimental

The sample preparation procedure (based on reference 7) ispresented in figure 1. (more details of clean up will bepublished soon). HPLC separation was carried out using anAgilent 1100 HPLC system with degasser, binary pump andautosampler, a Phenomenex column LUNA 50x2mm (3µm)with Security Guard column, and a gradient of water + 2mMammonium acetate (eluent A) and methanol (eluent B). A flowrate of 350µL and gradient profile (A/B) of 0 min 55/45, 5 min 10/90, 10 min 10/90, followed by re-equilibration wasused. A sample volume of 50 µL was injected.

I

16BiosystemsSolutions

a rapid and sensitive method for the identification and

quantification of CAP in milk and milk products

using LC/MS/MS has been developed

appl icat ions

Determinationof residues of

chloramphenicolin milk and milk products

using HPLC/MS/MS

Klara Oresnik, Milojka Bedek and Julija Bericnik-Vrbovsek,ERICo, Ecological Research & Industrial Cooperation, Velenje, Slovenia

Gabor Balizs, Federal Institute for Risk Assessment, Berlin, Germany

André Schreiber, Applied Biosystems, Darmstadt, Germany

Chloramphenicol

Figure 1.

Sample clean up for milkand milk powder samples

Figure 2.

Chromatogramcontaining 4 MRMs of Chloramphenicol(1µg/mL in milk)

BiosystemsSolutions17

Detection was performed on an API 3000™ LC/MS/MSsystem with TurboIonSpray® Source in negative ion mode andMultiple Reaction Monitoring (MRM). The following MRMtransitions (Collision Energy) were used for quantitation:321/152 (-24V), 321/257 (-12V), 321/194 (-16V), and 321/176 (-20V). Under these conditions, MRM 321/152was the most intense transition and, thus, used as quantifierwhile other transitions were used as qualifiers (figure 2).

The validation was done according to the criteria of theCommission Decision 657/2002/EC8.

The deviation of retention times between standard and sampleswere lower than 2.5% (the average retention time of standard2.54 min and 2.56 min of the milk samples).

A system of identification points must be used according to 657/2002/EC when mass spectrometric detection follows chromatographic separation. For the confirmation of substances listed in Group A of Annex I according to 96/23/EC9, like chloramphenicol, a minimum of 4 identification points is required. According to this definition,4 identification points earned using LC/MS/MS with 1 precursor and 2 product ions. The presented method detects4 product ions, thus, performance criteria for confirmation arefulfilled. Relative ion intensities are used to qualify results ofquantitation. The deviation of relative ion intensity in spikedmilk samples (vs. standard) was lower than 10%.

The calculated limit of decision (cca) was found at 0.03 µg/L,the detection capability (ccß) at 0.1 µg/L, and the recovery at 70% with a coefficient of variation of 5%. Coefficient ofcorrelation of > 0.99 for all 4 transitions were determined (7 points of calibration and 10 replicates).

Conclusion

This method is able to identify and quantify residues ofchloramphenicol in milk and milk products in lower ppb concentration range. Due to its high specificity the LC/MS/MS assay fulfils criteria of the EU regulation657/2002/EC.

References

1. D. Arnold and A. Somogyi: J. AOAC 68 (1985) 984

2. G. Balizs and D. Arnold: Chromatographia, 27 (1989) 489

3. P.Fürst, C.Krüger, H.A.Meemken, and W.Groebel: D. Lebensmittel

Rundschau 84 (1988) 108

4. P.J.Kijak: J. AOAC 77 (1994) 34

5. A.Pfenning, S.Turnipseed, J.Roybal, M.Madson, R.Lee and J.Storey:

U.S. Food and Drug Administration, Denver, LIB No.4294, 19 (2003)

6. A.Gantverg, I.Shishani and M. Hoffman: Analytica Chimica Acta 483 (2003) 125

7. V. Hormazabal, M. Yndestad: J. Liq. Chrom. & Rel. Technol.24 (2001) 2477

8. Community Decision 657/2002/EC

9. Council Directive 96/23/EC of 29 April 1996

INFO

Contact

usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]

For more information on:The API 3000 LC/MS/MS System enter: 107

To download related application notes, visit:http://www.appliedbiosystems.eu.com/mk/get/ABSMPDFREG

Related article can be found on: Page 10

This method is able to identify and quantify residues

of chloramphenicol in milk and milk products

in lower ppb concentration range

n February 2003, a mysterious illness resemblingpneumonia began to spread rapidly through differentparts of Asia. At first, it was thought that an outbreak of

bird flu virus, similar to the one that killed six people in 1997,was to blame1. However, the next month, the world wasintroduced to the name Severe Acute Respiratory Syndrome,or SARS and the mysterious disease quickly spread from Asia to six continents in a matter of weeks.

In fact, just a few months after a name was given to the mystery disease, researchers armed with products from Applied Biosystems and technologies had already developedthe first SARS diagnostic test, and identified key viral proteinsthat represent potential drug targets. Identification of the virusresponsible for SARS, and high-throughput DNA sequencingof the viral genome have helped researchers to betterunderstand the origins of the disease

SARS spread quickly

In late 2002 sporadic cases of a mysterious respiratory illness first began to occur in Guangdong Province, China.About three months later the disease had spread to severaldifferent countries through international air travel. At that time, about a dozen people – all of whom werebelieved to have contracted SARS from a GuangdongProvince doctor while they all stayed on the same floor of the Metropole Hotel in Hong Kong – left Hong Kong andfanned out to different parts of the world2. It then spread toSingapore, Canada, and Vietnam. And it was in Vietnamwhere the illness claimed the life of Dr. Carlo Urbani, a WorldHealth Organization (WHO) physician, who was the first oneto identify the outbreak while treating one of the first patientsstricken with SARS, a sick traveller from Hong Kong2.

I

18BiosystemsSolutions

The world was introduced to the name Severe Acute Respiratory Syndrome,

or SARS and the mysterious disease quickly spread from

Asia to six continents in a matter of weeks

appl icat ions

Applied Biosystemstechnology

arms researchers

by Mark SpringerBioBeat®

Online Magazinewww.biobeat.com

in the battle against

SARS

ABI PRISM 3100 Genetic Analyzer

ABI PRISM 7000 SequenceDetection System

GeneAmp PCR System 9700

Public health efforts reduce risk,

but danger remains

Today, thanks to global public health efforts and goodinfection control practices around the world, new SARS cases have disappeared, with only a few infections being reported since July 20033. However, the apparentdisappearance of SARS does not necessarily mean the disease will not strike again3.

Weekend discovery

In March 2003, Dr. Christian Drosten, Bernhard NochtInstitute for Tropical Medicine, Hamburg, Germany workednon-stop for an entire weekend to determine the geneticmakeup of the virus responsible for SARS from a cell cultureinfected with material from a SARS patient sample.

Based on the viral genetic sequences he discovered, he developed a real-time RT-PCR (reverse transcription-polymerase chain reaction) assay that was subsequentlyincorporated into the first SARS diagnostic test, a test that iscurrently being marketed by Abbott labs and distributedthrough its molecular diagnostics alliance with CeleraDiagnostics, a joint venture of Applied Biosystems and CeleraGenomics, two business groups of the Applera Corporation4.Developed into a commercial kit by the German companyArtus GmbH, and based on the technique of real-time PCR,the SARS diagnostic test detects the presence of the SARS viralgenetic material in patient blood and tissue samples.

The diagnostic test

The SARS diagnostic test is based on a real-time RT-PCR assay that uses probes and primers made from DNA fragments selected from regions of the SARS-associatedcoronavirus (SARS-CoV) genome. Researchers at the BernhardNocht Institute used an ABI PRISM® 3100 Genetic Analyzerand BigDye® Terminator sequencing reagents for all DNA sequencing reactions needed for development of primers and probes used in the real-time RT-PCR assay. The ABI PRISM® 7000 Sequence Detection System, also fromApplied Biosystems, automated many of the real-time RT-PCRassays used in the development of the diagnostic test5.

Test can confirm, but not rule out

"The only way to use the PCR diagnostic test is to confirmsuspected [SARS] cases, not to rule out the disease," explainsDr. Drosten. "If you find a positive signal, then you can saythat it's really SARS," says Dr. Drosten. "WHO asks you to usea second test for confirmation, but that's usual laboratoryprocedure that you would also do with many other diseases toconfirm a positive PCR result with a second PCR test."

Sequence detection systems help

to quantify SARS virus

In addition, the SARS diagnostic test also measures differentlevels of expression of the viral genetic material in differentkinds of clinical specimens.

When the researchers quantified amounts of viral RNA byperforming a real-time RT-PCR assay, they found answers tomany of their questions. Real-time RT-PCR assays revealedsignificant disparity in the concentration of SARS-associatedcoronavirus in different kinds of samples containing viralgenetic material5.

For instance, sputum samples showed much higherconcentrations of viral RNA than did serum samples5.

"The problem is that the clinicians are a little bit afraid oftaking these samples of deep respiratory materials from SARSpatients, because when you have a SARS patient cough deepfrom his lungs, he [or she] will produce quite a lot of virus inthe air that will produce aerosols and put the clinicians at riskof becoming infected," says Dr. Drosten.

Positive past experience dictated choice

of Applied Biosystems technology

To detect the presence and amount of virus detected indifferent clinical specimens, researchers used fluorescently-labelled coronavirus-specific probes and primers, and automated many of the real-time RT-PCR assays using the 7000 system.

Dr. Drosten is a long-time user of sequence detection systemsfrom Applied Biosystems and his lab is equipped with both a7700 system and a 7000 system, instruments that he regularlyuses to automate RT-PCR assays.

"We use the 7000 system for a lot of our routine assays that areoperated by technicians," he continues. "It's very easy to use.It's quite easy to evaluate the data that you collect."

"Another reason [that we use Applied Biosystems sequencedetection systems] is that the thermal cycler inside this systemis one of the most renowned cyclers, the GeneAmp® PCRSystem 9700. And, in the previous model, the 7700 system,the GeneAmp® PCR 9600 system, a thermal cycler that hasbeen in operation for about 15 years. So, there is really reliabletechnology that you have as a basis for these machines," Dr. Drosten notes.

Virus identified through random

amplification approach

Having only part of the genomic sequence of the virus, the researchers identified the virus by randomly amplifying,and then sequencing fragments of the virus genome obtainedfrom tissue cultures and supernatant.

Above.

Dr. Christian Drosten, Bernhard Nocht Institute

for Tropical Medicine,Hamburg, Germany

BiosystemsSolutions19

“The first thing that I did was a cross titration to

look at different combinations of primers.

After that, we optimised the reagents.“

Page 20

INFO

For more information on:BigDye TerminatorSequencing Reagentsenter: 108

For more information on:ABI PRISM 3100 GeneticAnalyzer enter: 109

For more information on:Sequence DetectionSystems from Applied Biosystemsenter: 110

For more information on:Thermal Cyclers from Applied Biosystemsenter: 111

Related article can be found on:Page 38

Contact

usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]

20BiosystemsSolutions

To design the primers for use in the real-time RT-PCR assay, Dr. Drosten used Primer Express® Software from Applied Biosystems. He sought the best possible combinationof forward and reverse primers for the assay.

"The first thing that I did was a cross titration to look atdifferent combinations of primers. After that, we optimisedthe reagents."

Researchers identify mystery virus

as a coronavirus

A team of researchers led by Dr. Malik Peiris, and hiscolleague Dr. K.Y. Yuen, both from the Department ofMicrobiology at the University of Hong Kong discovered acluster of viral particles lurking amidst a group of dying cells.They recognised the crown-like spherical membranessurrounding each viral particle as the trademark signature of a family of viruses known as coronaviruses, perhaps bestknown for being one of the virus families responsible for the common cold2.

The coronaviruses are RNA viruses which are less stable thanDNA viruses and tend to mutate more frequently6.

Simultaneously, Dr. Drosten and Dr. Malik Peiris embarkedupon different paths of research, only to both arrive at the same conclusion that the culprit behind the SARS epidemicwas a new kind of coronavirus, one never before known to infect humans.

Applied Biosystems 3100 System chosen

for critical sequencing steps

Following the RT-PCR procedure, Dr. Drosten's teamdetermined the nucleotide sequence of amplified DNAfragments. For this, they used BigDye® terminator sequencingreagents and a 3100 system from Applied Biosystems toautomate the sequencing reactions5.

It was determined that the SARS-CoV contained nucleotidesequences different from those found in any other knowncoronavirus. In fact, comparative sequence analysis studiesshowed that genes from the SARS-CoV shared only a partial DNA sequence similarity with other knowncoronaviruses recorded from previous investigations of both humans and animals5.

Knowledge of SARS proteins provides clues

for drug researchers

Other SARS-CoV proteins of interest to drug developers are those that are part of the RNA replication complex of the virus, enzymes responsible for operating the machinerythat enables the virus to copy its genetic material once inside a host cell.

For example, similarities in the structure of the viral mainproteinase (also called 3CLpro), an essential component of theSARS-CoV replication machinery, to a protein of similarfunction in rhinoviruses, viruses that can cause the commoncold, attracted the attention of researchers at the University ofLübeck, in Germany7.

These researchers found that it might be possible to modify an available cold virus drug so that it inhibits the functioningof the SARS-CoV proteinase, an enzyme that plays a key role in controlling the replication of SARS-CoV. The structureof the SARS-CoV proteinase resembles the structure of a cold virus protein previously found to be inhibited byrhinovirus inhibitors7.

Where did SARS come from and where is it going?

To investigate the origins of the SARS virus, scientists have already studied variations in the amino acid sequence of components of the spike protein. Or, if they can locate a similar virus in animals, they may be able to determineexactly what mutation permitted it to infect humans for the first time8.

"There are some parallels for coronaviruses in animals," notes Dr. Drosten. "For example, there is a kind of pigcoronavirus that once caused very high mortality rates in pigs, and then it lost some genomic fragment."

A constantly changing genome increases the chances that avirus will find new ways of infecting individuals which isfrustrating for researchers who attempt to develop a singlevaccine for it. Although there are concerns that as mutationsmount, odds also increase that an even more virulent SARS-CoV strain will emerge.

If another outbreak of SARS does occur, the rapid response of research scientists to the initial outbreak of the disease,coupled with steady advances that have been made inunderstanding the nature of the virus will most likely make it easier in the future to both identify and combat the spreadof the disease.

References

1. Bradsher, K., "Man's Death of 'Bird Flu' in Hong Kong Raises Fears," The New York Times A7 (February 21, 2003)

2. Pottinger, M., Cherney, E., Naik, G., Waldholz, M., "How Global Effort Found SARS Virus in Matter of Weeks," The Wall Street Journal A1 (April 16, 2003)

3. Enserink, M., "The Big Question Now: Will it Be Back," Science 301:299 (July 18, 2003).

4. "Abbott Laboratories Enters Agreement with Artus GMBH for SARS Diagnostic Test," Press Release, Abbott Laboratories, Abbott Park, Illinois (May 15, 2003)

5. Drosten, C. et al., "Identification of a Novel Coronavirus in Patientswith Severe Acute Respiratory Syndrome," The New England Journal of Medicine 348(20): 1967-1976 (May 15, 2003).

6. Liu, E., "Science and Societal Challenges: SARS, Singapore, and itsBiomedical Research Institute," presentation at International Congress of Genetics meeting in Australia (July 10, 2003)

7. Anand, K., et al., "Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs," Science 300: 1763-1767 (June 13, 2003).

8. Pottinger, M., Regalado, A., "Mutating Virus Keeps Scientists Chasing SARS," The Wall Street Journal B1 (May 09, 2003)

Above.

CoronavirusAn electron microscopicphoto of the Frankfurt,

Germany strain of the SARS-associated

coronavirus (SARS-CoV). Photo captured byProfessor Herbert

Schmitz of the Bernhard Nocht Institute

for Tropical Medicine. Permission for use

of photo granted by Dr. Christian Drosten.

BiosystemsSolutions21

Project leader Dr Jessica Zucman-Rossi explained:

“The aim of our research is to understand the molecular

events and characterise the pathways involved with the

development of hepatocellular carcinomas and, from there,

to identify new diagnostic and prognostic markers.

Following the identification of a number of differentially

expressed genes, we use TaqMan® Gene Expression Assays

to analyse their expression in a series of 100 hepatocellular

carcinomas, benign tumours and corresponding

non-tumour liver tissues.”

“It was important for us to find a robust system that could

cope with the high workload this involved and would allow

us to standardise our procedures.

The TaqMan® Low Density Array looked very promising

in both of these respects and we were impressed that

Applied Biosystems offered such a large range of pre-loaded

and validated assays that could be shipped to us

quickly. We have found the quality of the gene

database and annotations to be very high and like the

fact that the human assays can be complemented

with mouse assay sets if required.” Jessica concluded:

“The Applied Biosystems Application Support team helped

us to design the TaqMan Gene Expression Assays for

our tumour series and the experimental protocols to go

with it. They were also on hand to help us validate and

interpret the first results we obtained.”

A

For more information on:TaqMan GeneExpression Assays enter: 112

For more information on:TaqMan Low DensityArray enter: 113

For more information on:The articles mentioned inthe references enter: 114

Related article can be found on:Page 38

INFO

appl icat ions

Above

Dr. Jessica Zucman-Rossi, Centre d’Etude du Polymorphisme Humain

(CEPH), Paris

High-Throughputgene expression systemfor a liver carcinoma research project at CEPH, Paris

“The TaqMan Low Density Array looked very promising and we were impressed that Applied Biosystems

offered such a large range of pre-loaded and validated assays that

could be shipped to us quickly”

group of Inserm scientists at the Centre d'Etude du Polymorphisme Humain (CEPH) in Paris turned

to Applied Biosystems when it needed high throughput equipment to analyse differentially expressed

genes in hepatocellular tumours.

References1. J Clin Endocrinol Metab. 2004 Mar;89(3):1476-80.

Hepatocyte nuclear factor-1 alpha gene inactivation:cosegregation between liver adenomatosis and diabetes

phenotypes in two maturity-onset diabetes of the young(MODY)3 families. Reznik Y, Dao T, Coutant R,

Chiche L, Jeannot E, Clauin S, Rousselot P, Fabre M, Oberti F, Fatome A, Zucman-Rossi J,

Bellanne-Chantelot C.

2. Pathol Biol (Paris). 2004 Mar;52(2):60-2. Genetic alterations in hepatocellular adenomas

Zucman-Rossi J. [Article in French]

3. Nat Genet. 2002 Oct;32(2):312-5. Epub 2002 Sep23. Bi-allelic inactivation of TCF1 in hepatic

adenomas. Bluteau O, Jeannot E, Bioulac-Sage P,Marques JM, Blanc JF, Bui H, Beaudoin JC, Franco D,

Balabaud C, Laurent-Puig P,Zucman-Rossi J.

During the last year, Applied Biosystems Integrated Software Solutions

(ISS) team has been collaborating with the Biomedical Proteomics

Research Group (BPRG) of the Geneva University, lead by Prof. Denis F.

Hochstrasser, and the Proteome Informatics group of the Swiss Institute

of Bioinformatics to provide a LIMS solution for laboratories of the

BPRG and the proteomics platform of the Medical faculty of

Geneva University.

Think with the end in mind…

A number of challenges were faced before delivering a successful LIMS project:

» Ability to build dynamic workflows. In leading edge research

laboratories, the experiments are often designed during the total

experimental process, based on the intermediate results.

» Availability of specialised workflows for Molecular Scanning,

1DE/2DE master gel for best practices collection and ELISA,

immuno blots SELDI when using classical LC and gel-based

protein identification workflows (1D-SDS, 2D PAGE, uni- and

multi- dimensional mLC, MALDI and ESI MS or MS/MS).

» Robust extensible data management system (SQL*LIMS®)

to enable the Proteome Informatics group of the Swiss Institute

of Bioinformatics to support their infrastructure. A high level

of quality in the collected data and very detailed sample tracking

through the analytical process, from preparation to final protein

identification, is mandatory.

» Flexible workflows design procedures to support the Medical

faculty for the Geneva University to provide a service for their

external customers.

22BiosystemsSolutions

appl icat ions

cientists involved in protein expression analysis today

have more and more to deal with due to complex

combinations of basic workflows and massive amounts of

raw data. Increasingly they need better data management and

analytical tools for sample tracking and visualisation of the

meaning behind the raw data. A growing number of scientists

are now turning to Laboratory Information Management Systems

(LIMS) to remove any obstacles in productivity and streamline

their operations.

Sresearch environment

in a leading edge

Scientists involved in protein expression analysis today have more and more to deal

with due to complex combinations of basic workflows and massive amounts of

raw data. Increasingly they need better data management and analytical tools

for sample tracking and visualisation of the meaning behind the raw data

ProteomicsSolution

The integrated solution

To fulfil the project requirements, Applied Biosystems ISS

team has provided a solution leveraging the Life Science

LIMS application, from the SQL*LIMS family, and the ISS

Proteomics Component suite (see figure 1).

» Operating flexibility is provided by SQL*LIMS, which allows

users to design their own workflows.

» Easy extensibility is supported by an open interface using

built-in APIs and add-on mechanisms. MS instruments and

third-party tools for gel image and protein identification can

be easily integrated allowing direct transfer of real-time,

bi-directional data in or out of the LIMS system.

» Usability is guaranteed by application-specific interfaces for

most of the basic sample operations (aliquoting, spot picking,

MS data loading, protein identification etc.).

» Enhanced data access features are also made available,

providing complete hierarchical overview of studies,

parent-child sample relationships & protein validation results.

Along with a high level of integration with the latest generation

of MS instruments, including the 4700 Proteomics Analyzer,

and search engines, this solution allows scientists to leverage a

results driven strategy.

Future and Challenges

The BPRG and the proteomics platform of the Medical

faculty of the Geneva University now have an up-to-date

LIMS system for their proteomics laboratories. Their next

challenge is to educate users about the importance of a

LIMS system as a unique repository of their data. LIMS will

become the elective source of data for any further

investigations and a milestone into the ‘biological stream’,

with sequencing and genomics for upstream and

transcriptomics for downstream.

BiosystemsSolutions23

INFO

Figure 1

SQL*LIMS removedobstacles in productivityfor scientists at BPRG, in Geneva University

AcknowledgementsP. Binz1,2, S.Paesano3, P. Zanini4, A. Scherl3, C. Hoogland1, L. Allard3,C. Zimmermann3, O. Carrette3, J. Sanchez3, S. Borella4,R. Castelnovo4, R. Appel1,2,5, D.F. Hochstrasser2,3,5

1. Swiss InstituteBioinformatics

2. Faculty of Medicine, Geneva University

3. Biomedical Proteomics Research Group, Geneva University

4. Applied Biosystems, Italy

5. University Hospital, Geneva

LIMS will become the elective source of data for any further investigations

and a milestone into the ‘biological stream’, with sequencing and

genomics for upstream and transcriptomics for downstream

For more information on:LIMS in ProteomicsResearch enter: 115

For more information on:LIMS in GenomicsResearch enter: 116

Introduction

he renin-angiotensin system plays an important role in regulating blood volume, arterial pressure, and cardiac and vascular function. Management of this

pathway has become very important in the treatment of highblood pressure and heart failure. Angiotensin II is a smallpeptide biomarker that is used to monitor vascular health at the endocrine level. Angiotensin I is metabolised toAngiotensin II by the Angiotensin converting enzyme (ACE).Many treatments for high blood pressure involve prescribingACE inhibitors to block the processing of Angiotensin I byAngiotensin-converting enzyme. This lowers blood pressure byreducing the circulating levels of Angiotensin II.

Experimental

An LC/MS/MS assay was developed to quantify Angiotensin IIpeptide levels in plasma using the 4000 Q TRAP system. The assay was performed using the TURBO V™ Source at very high LC flow rates (700 µL/min) to maximise robustness and throughput without sacrificing sensitivity. Using a 2x50mm C18 column (Targa, Higgins Analytical) and a short gradient (5-75% acetonitrile in 0.1% formic acid in two minutes), the total assay time was under fiveminutes. The Angiotensin II peptide eluted from the column in a narrow peak at a retention time of 1.0 minute.

Because of the high sensitivity and specificity provided by Multiple Reaction Monitoring (MRM), this MS/MS triple quadrupole scan mode was used for the assay. At high flow, two MRM transitions were monitored for Angiotenisin II (349.83+ ➞ 371.21+ and 349.83+ ➞ 136.11+). Both Q1 and Q3 were set to transmit a ~1 Da window centred on the masses specified. Using the QuantitativeOptimisation tool in Analyst® Software 1.4, instrumentparameters were automatically optimised for each MRM transition.

Angiotensin II peptide (Sigma) was used to generate a standardconcentration curve using the method described. Rat plasma(Pel-Freez Biologicals) was prepared using standard organicprecipitation techniques to remove the protein content, leaving peptides, metabolites, etc. in solution. The plasma was mixed with acetonitrile in a 1:4 ratio. The supernatant wasdried down, and then re-suspended in 10% acetonitrile / 0.1 %formic acid.

Results from the MRM assays were automatically processedusing the Quantitation Wizard in the Analyst® Software 1.4.Summing and integration of the MRM peaks, calculation of thestandard concentration curves and statistics were automaticallyperformed and the results report generated.

To investigate the optimal level of sensitivity, the assay was alsoperformed at nanoflow rates (250 nL/min) using theNanoSpray™ Source. Using a 0.075 x 150 mm C18 column(Vydac) at 250 nL/min and a slightly longer gradient (5-75% acetonitrile in 0.1% formic acid in twenty minutes),the total assay time was 30 minutes. At low flow rates, the doubly charged precursor ion was predominant so theMRM transitions 523.82+ ➞ 263.11+ and 523.82+ ➞ 784.61+

were monitored and summed.

Results

Limit of Quantitation of Angiotensin II at High Flow Rates A standard curve was run to determine the mass spectrometerresponse for this peptide under the assay conditions definedabove. The standard peptide was run sequentially at increasingconcentration. The curve was determined to be linear over four orders of magnitude (0.160 – 2550 fmole on column, R= 0.9994, linear regression, 1/x weighting, see inset, figure 1).The peptide could be accurately detected and quantified downto 160 amole on column using this assay with a signal to noise ratio (S/N) of 30:1. The MRM trace for 160 amole ofAngiotensin II peptide on column is shown in figure 1.Typically, S/N ratios of 10/1 define the lowest limit for accuratequantitation (LOQ), suggesting that limits down to ~50 amoleon column would be achievable and quantifiable by thismethod. It was also determined that summing the two MRM transitions together improved the S/N ratios obtainedand therefore the limits of quantitation.

24BiosystemsSolutions

The identification of the proteins is based on the mascot score

of the peptide mass fingerprint and of the daughter

ion spectra of the tryptic peptides

appl icat ions

INFO

For more information on:4000 Q TRAP Systementer: 117

Related article can be found on:Page 14

AcknowledgementsChristie Hunter,Louisette Basa,Applied Biosystems,USA

Detecting aPeptide Biomarker

for Hypertension in PlasmaPeptide Quantitation Using the 4000 Q TRAP™ System

T

Table 1.

Limits of Quantitation(LOQ) obtained forAngiotensin II at variousflow rates.

BiosystemsSolutions25

Flow Rate Assay LOQ S/N Dynamic Range(µL/min) Time (min) (amole)

700 4.5 160 30/1 0.16 to 255 fmole4 orders

0.250 32 16 13/1 0.016 to 51 fmole4 orders

Figure 1.

Standard concentrationcurve of Angiotensin II.MRM LC trace (sum of349.83+ ➞ 371.21+ and349.83+ ➞ 136.11+) showsthe limit of detection of 160 amole on column (S/N of 30) at 700 µL/min. Inset shows the linearstandard concentrationcurve (R=0.9994).

Figure 2.

Standard concentrationcurve of Angiotensin II.MRM LC trace shows the limit of quantitation of 16 amole on column(S/N of 13) at 250 nL/min.

Figure 3.

Endogenous AngiotenisinII detected in rat plasma.The equivalent of 20 µL of protein-precipitated ratplasma was injected oncolumn and the area of the peak compared to thestandard curve in Figure 2.6.6 fmole of angiotesin IIwas detected on column,which corresponds to acirculating level of 330amole Angiotensin II per µL of plasma. (MRM transitions shown:red trace 349.83+ ➞ 371.21+

|and blue trace 349.83+

fi 136.11+).

Limit of Quantitation of Angiotensin at Low Flow RatesTo assess the levels of detection and quantitation at nanoflowrates, the standard concentration curve was also measured usingthe NanoSpray™ Source at 250 nL/min on the 4000 Q TRAPsystem. For this assay, the limit of quantitation of AngiotensinII peptide was 16 amole on column with a S/N of 13/1 (figure 2). Ten fold greater sensitivity was achieved, but atlonger assay times of 30 minutes.

Detection of Endogenous Angiotensin II in Rat PlasmaAfter the standard curve was generated, a sample of plasma was run under identical high flow assay conditions. A strong peak was seen at the same retention time and wasdetermined to have a concentration on column of 6.6 fmole(figure 3). This calculates back to be an initial plasmaconcentration of 330 amole of Angiotensin II per mL of plasma(0.35 ng peptide / µL plasma). This peak can be putativelyattributed to the presence of Angiotensin II in rat plasmabecause the retention time of the LC peak is similar to thestandard curve and because the two fragment ions monitored by MRM are co-eluting (figure 3, red and blue trace).

Summary of Quantitation Results

The Multiple Reaction Monitoring experiment of the 4000 Q TRAP system allows for extremely sensitive andselective detection of specific biomarkers in protein-precipitatedplasma. The efficiency of the TURBO V™ Source at high flowrates allows for rapid and robust assays to be developed whileretaining high sensitivity. Optimal sensitivity is achieved atnanoflow rates but at a much lower throughput.

The unique combination of a triple quadrupole and ion trapfunctionality in the 4000 Q TRAP System allows forconfirmatory MS/MS information to be obtained at highsensitivity during the quantitation assay for higher confidenceresults in complex mixtures.

Contact

usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]

Genome-wideExpression Profilingwith the

Applied BiosystemsExpression Array System

Abstract

xpression profiling1 of over 30,000 genes in paired normal and breast tumour tissue on the Applied Biosystems Expression Array System revealed

numerous genes, which were differentially expressed inprimary and metastasis tumours, compared to normal tissuefrom the same patient. The study included biological andtechnical replicates. Changes in the expression levels werevalidated by real-time PCR with TaqMan® probe based assays.The study demonstrates the use of microarrays for genome-wide screening for gene expression, in combination withquantitative real-time PCR for validating and extending resultsfor genes of interest.

Methods

Two biological and two technical replicates were analysed forthree different disease states. Total RNA from normal, primaryand metastasis tumours of two patients was isolated and labelledwith Digoxigenin using the Applied Biosystems RT-IVT Kit.The labelled cRNA was hybridised to the Applied BiosystemsHuman Genome Survey Micro Array and array duplicates were processed for each of the three tissues for each of the two patients. Following hybridisation and washing,chemiluminescent detection assays were carried out on thearrays, the resulting signal was read on the Applied BiosystemsChemiluminescent 1700 Microarray Analyzer.

Results and Discussion

Scatter plots of the technical replicates showed a goodcorrelation for the same sample of the same patient (figure 1A). The correlation was reduced when comparingbiological replicates of the same disease status in the twopatients (figure 1B), while the correlation was further reducedwhen comparing different disease conditions in the samepatients (figure 1C). While the second result points atdifferences in the gene expression of the individuals, the low correlation between disease conditions in the sameindividual indicates substantial gene expression changesbetween disease stages.

Using ANOVA test P<0.01 for all 12 arrays identified 2,058genes with significant differences in gene expression level and2-D hierarchical clustering analysis was performed usingGeneSpring® software (Silicon Genetics, Redwood City, CA,USA) (figure 2A). The primary tumour expression profile from patient 1 resembles more the metastasis pattern, while that of primary tumour sample from patient 2 lookedmore like the expression profile in normal tissue.

The gene annotation database and the PANTHER™

classification system for molecular function and biologicalprocess information, which is an integral part of the ExpressionArray System, allowed rapid identification of 200 genes which are involved in various signal transduction pathways(Expression profiles in figure 2B). Thirty genes withcharacteristic clustering (figure 2C) were validated with real-time PCR and TaqMan probes.

E

26BiosystemsSolutions

INFO

For more information on:Applied BiosystemsExpression Array Systementer: 118

the low correlation between disease conditions in the same

individual indicates substantial gene expression

changes between disease stages

appl icat ions

AcknowledgementsKelly Li, Irene Lui and Gary P. Schroth,Applied Biosystems,USA

A group of genes could be determined which were not detected innormal tissue but which show markedly increased expression in thecancerous tissues across all four samples for the same diseasecondition. Such genes could potentially serve as markers forchanges in disease.

A list of ‘unknown’ genes showed significant changes in Expression levels (p<0.001), based on expression data from theApplied Biosystems arrays. PANTHER classification allowedassigning molecular function and biological processes to many ofthese ‘unknown’ genes, based on structural classification of theunknown genes and comparison of these structural motifs withthose of well-characterised gene products. This approach could beuseful to determine potential markers indicating changes in disease.

Expression changes of 30 genes detected by microarrays wereconfirmed by real-time PCR using Gene Expression Assays fromApplied Biosystems across the 3 disease conditions and all 4 replicates. For microarrays, gene expression fold changes werecalculated as a ratio of tumour sample to matched normal tissueafter median global normalisation. For TaqMan data, fold changewas calculated using relative quantitation, or ∆∆Ct (delta delta Ct)method, where ∆∆Ct = ∆Ct tumour – ∆Ct normal, and ∆Ct isGAPDH normalised threshold cycle Ct. Fold changes seen byTaqMan assays and by the Expression Array System are similar.

Conclusion

Statistically significant gene expression changes were identifiedwhen comparing normal versus cancerous breast tissues on theApplied Biosystems Expression Array System. The utility of thearray system in the identification of potential biomarkers and novelgene functions using the PANTHER classification system wasdemonstrated. TaqMan probe-based assays confirmed expressionchanges observed on the microarray. Genome wide expressionscreening using the Expression Array System can be easily linkedwith quantitative real-time PCR for validating results for genes ofinterest. These TaqMan assays can also be used to extend the arrayobservation across many new samples.

References

1. Genome Wide Expression Profiling of Paired Cancerous and Normal

Breast Tissue. Conference poster by Kelly Li, Irene Lui and Gary P.

Schroth, presented at the 6th Joint Conference of the American

Association of Cancer Research and the Japanese Cancer Association,

January 25 – 29, 2004, in Waikoloa, Hawaii.

Figure 1.

Scatter Plots of Replicates.

A.Technical replicates comparing the samesample condition from the same patient.

B.Biological replicates comparing the same

disease condition in two different patients.

C.Comparison of primary tumour tissue with

metastasis tissue from the same patient.

BiosystemsSolutions27

Genome wide expression screening using the Expression Array

System can be easily linked with quantitative real-time

PCR for validating results for genes of interest

Figure 2.

2-D hierarchical clustering analysis of expression profiles on the Human Genome Survey Microarray. Red and blue colour represents high and low expression respectively and yellowrepresents unchanged expression. All experimental and biological replicates are clustered together. All arrays for normal (N), and metastasis (MT) tissue were clustered into their owngroups, primary tumour (PT) are associated with the pattern of normal or metastasis profiles they resemble more. A) 2,508 genes clustered into two main clusters. The top clusterindicates increasing expression and the bottom cluster shows decreasing expression of genes in tumours relative to normal tissue. B) Expression patterns of 200 out of 2,508 genes,which are involved in signal transduction pathways as classified by the PANTHER system. C) Cluster analysis of 30 genes used in TaqMan validation studies.

28BiosystemsSolutions

Abstract

poptosis is involved in almost every physiologic

and pathogenic process in the body. Knowledge of the

molecular mechanism of apoptosis has revealed

new approaches for identifying small-molecule drugs that may

effectively treat diseases including cancer, autoimmunity,

stroke, and osteoporosis. In addition, it is important to

determine whether lead compounds are cytotoxic in order to

eliminate them early in the drug discovery process.

An apoptosis and a necrosis assay have been developed

for use with the Applied Biosystems 8200 Cellular Detection

System. The apoptosis assay uses Annexin V labelled with

a red laser compatible dye to identify apoptotic cells,

while the necrosis assay uses a DNA binding dye that is

excluded from live cells. Both assays utilise CentriRed®

stain for enumeration of the cells present in the scan area.

The 8200 system itself uses a red laser to scan 96-, 384- and

1536-well plates and quantifies cell- or bead-associated

fluorescence using a proprietary algorithm. These assays are

mix-and-read and can be performed on either adherent or

suspension type tissue culture cells.

A

appl icat ions

Assessment of Apoptosis andlead compound CytotoxicityUsing the Applied Biosystems 8200 Cellular Detection System

An apoptosis and a necrosis assay have been developed for use

with the Applied Biosystems 8200 Cellular Detection System

Introduction

Apoptosis (programmed cell death) is a regulated process

that multicellular organisms use to destroy superfluous cells.

The apoptotic cell bodies are phagocytosed prior to cell

lysis with little or no immune response, thereby eliminating

activation of the detrimental inflammatory process.

Apoptosis involves two known pathways that can be termed

extrinsic and intrinsic. The extrinsic pathway is stimulated by

protein or small molecule ligands that bind to cell surface death

receptors (e.g. FasL) and activate the upstream caspases directly.

The intrinsic pathway is induced by cellular stresses and refers

to mitochondrial processes that involve the BCL-2 family of

proteins, causing the release of cytochrome C and leading to the

activation of downstream caspases1. Both pathways converge

with the activation of caspase-3 and induce morphological

changes such as cytoplasmic shrinkage, active membrane

blebbing and chromatin condensation. Biochemical changes

include the activation of caspases, the externalisation of

phosphatidylserine and DNA fragmentation. Annexin V

binding to phosphatidylserine exposed to the outer leaflet of the

plasma membrane has been shown to be a reliable early marker

for cells undergoing apoptosis2. Recombinant Annexin V

labelled with a red laser dye and CentriRed stain have been

combined to generate a 2-colour apoptosis assay that can be

performed in 96- or 384-well plates (figure 1A). We have used

human hepatocellular carcinoma (HCT116) cells treated with

staurosporine as a model system for apoptosis. Staurosporine is

a potent inhibitor of protein kinase C that induces apoptosis in

cultured cells. CentriRed nucleic acid binding dye stains both

viable and dead cells. It is used to enumerate all of the cells in

the scan area so that the data can be presented as the percentage

of cells within the scan area that are binding Annexin V.

Alternatively, cells that undergo necrosis as a response to the

accumulation of toxic compounds display rapid degradation,

increases in plasma membrane permeability, swelling of the

mitochondria and a subsequent host immune response.

One hallmark of necrosis is the ability of certain dyes such as

trypan blue and propidium iodide to pass through the

membrane and stain the cell.

TOTO®-3 dye is a high affinity DNA labelling red laser

dye suitable for use on the 8200 system that stains only dead

cells with compromised plasma membranes. In this assay,

TOTO-3 dye is used to identify necrotic cells and CentriRed

dye is used to enumerate all cells in the scan area and the result

is presented as a percentage of the total.

Materials and Methods

Cell culture and Induction of Apoptosis: HCT-116 cells(Colorectal Carcinoma, ATCC) were plated in 100 µl complete media (RPMI-1640, 10% FBS and 1% Penicillin/Streptomycin) at 10,000 cells per well in a 96-well FMAT®

plate. The cells were plated 24 hours prior to drug treatment to allow them to attach firmly to the bottom of the well. A dose response curve for staurosporine (Sigma) treatment was performed.

FirstStaurosporine (214 µM in absolute ethanol) was dilutedto a final concentration of 6 µM (6000 nM) in completemedia.

Second10 serial 3-fold dilutions were performed to produce all 11 points on the curve with the last column containingno staurosporine as a control.

Third100 µl of media or media plus staurosporine was added to each well (6 replicates) and the final volume in every assay well contained 200 µl.

NOTE: For the hit plate assay, 6 individual wells were treated with 3 µM staurosporine and the remaining wells wereuntreated. Cells were incubated overnight (18-20 hours) in a37°C, CO2 incubator

BiosystemsSolutions29

Apoptosis (programmed cell death) is a regulated

process that multicellular organisms

use to destroy superfluous cells

Above: Figure 1.

A) Greyscale imagescaptured with an 8200system assay. In thehealthy control wells, CentriRed stain bindingis visible (images 1 and3). In the sample wellsundergoing apoptosis, the characteristicblebbing can bevisualised in the image(image 2). In wellsshowing necrosis, the cells stainpredominantly withTOTO-3 (image 4).CentriRed stain doesnot bind to apoptotic/necrotic cells that havelost their nucleic acidsinto the surroundingmedia. Therefore, in thewells treated with 3 µMstaurosporine (wells 2and 4), the binding is represented by Dye-labelled Annexin V2

or TOTO-34 only.

B) Graphs showing thepercentage of apoptosisor necrosis versus theconcentration of staurosporine (nM). The percentage iscalculated by dividingAnnexin V or TOTO-3binding events by the total number ofevents. This assay wasperformed in a 96-wellplate and each conditionhad replicates of 6. The estimated IC50 is 35 nM staurosporine for Annexin V bindingand 1 mM for TOTO-3dye binding.

Alexa® 647- Annexin V

TOTO® -3

AAnnexin V-Alexa®647 Annexin V-APC TOTO®-3

B

Page 30

Per

cen

t ap

op

tosi

s/n

ecro

sis

1 2

3 4

30BiosystemsSolutions

Note: For the hit plate assay, 6 individual wells were treated with3 mM staurosporine and the remaining wells were untreated.Cells were incubated overnight (18-20 hours) in a 37°C, CO2 incubator.

Red laser dye-labelled Annexin V binding assay: CentriRedstain (Applied Biosystems: 10 µM stock in anhydrousmethanol) and Annexin V-Alexa Fluor® 647 (MolecularProbes) were added to 5x Annexin V binding buffer (12.5 mMCalcium Chloride, 140 mM Sodium Chloride, 10 mM Hepes)at concentrations of 15 nM and 1:800 respectively. 50 µl of the above dye mixture was added to each well of thestaurosporine-treated 96-well plate so that the final volume was250 µl. The addition of both CentriRed stain (final 3 nM) and Annexin V-Alexa Fluor 647 (final 1:4000) allows 2-colouranalysis and calculation of the percentage of cells in the scanarea that are binding to the Annexin V protein. For theAnnexin V-APC assay, Annexin V-APC (Molecular Probes)replaced Annexin V-Alexa Fluor 647 at a final concentration of 1:4000.

TOTO-3 dye binding assay: CentriRed stain (AppliedBiosystems) and TOTO-3 dye (Molecular Probes) were added to 5x Annexin V binding buffer at final concentrations of 15 nM and 75 nM respectively. 50 µl of the above dye mixture was added to each well so that the final volume was 250 µl. The addition of both CentriRed stain (final 3 nM) and TOTO-3 dye (final 15 nM) allows 2-colour analysis and calculation of the percentage of cells in the scan area that are stained with TOTO-3 dye. For both assays the plates were incubated in the dark at room temperature for 1-4 hours prior to scanning.

Results and Conclusions

It is believed that the ability of chemotherapeutic drugs toinduce apoptosis increases their efficacy3. Therefore, the abilityto rapidly screen synthetic or natural compound libraries forsmall molecule drugs that induce apoptosis is useful for drugdiscovery efforts in oncology. In addition, defects in theapoptotic pathway may be involved in other disordersassociated with cell accumulation such as autoimmunity andinflammation. The 8200 system apoptosis assay is based on thebinding of dye-labelled Annexin V to phosphatidylserine thatis exposed to the outer surface of the cell membrane in cellsundergoing apoptosis. The IC50 for staurosporine-inducedapoptosis in this assay has been determined to be 35 nM which is consistent with literature values. This assay hasbeen shown to be robust and reproducible. We also show thatTOTO-3 dye can be used in the 8200 system to measure theamount of necrotic cells present in an assay well. The IC50 forstaurosporine-induced necrosis has been determined to be 1 mM. This assay can be used in addition to the Annexin Vbinding assay to gain more information about whether the testcompound also causes necrotic cell death. In addition to thenumerical data reported, grey scale and pseudocolour images are generated that enable well-by-well analysis of hits to observe the state of the cells that are identified asapoptotic or necrotic.

The 8200 system is an excellent tool for quantitative analysisof fluorescent molecules bound to the surface of tissue culturecells because they have minimal autofluorescence from the 633HeNe laser4. The 8200 system uses macroconfocal technologyto collect data from a 1mm2 area on the bottom of the well with an approximately 100 micron depth of focus. Cell-associated fluorescence is collected by two PMTs (PMT1,650-685; PMT2, 685-720 nm) and allows the deconvolutionof two different red laser dyes run concurrently in mix-and-read format. Multiple 96-, 384- or 1536-well plates can beloaded onto the automated plate handler allowing analysis ofthousands of compounds per day. Cell-based receptor-ligandbinding and hybridoma screening assays, and bead-basedimmunoassays can also be performed on the 8200 system.

References

1. Reed J.C.and Tomaselli, K.J. Drug discovery opportunities from

apoptosis research. Curr Opin Biotechnology, 2000 6:586-92.

2. Van Engeland, M., Nieland, L.J., Ramakers, F.C., Shutte, B.,

Reutelingsperger, C.P. Annexin V-affinity assay: a review on an

apoptosis detection system based on phosphatidylserine exposure.

Cytometry 1998; 31(1):1-9.

3. Tamm I., Schriever, F., Dorken, B. Apoptosis: implications of basic

research for clinical oncology. The Lancet-Oncology 2001; Vol 2:33-42.

4. Lee J.Y., Miraglia S, Yan X, Swartzman E, Cornell-Kennon S,

Mellentin-Michelotti, J, Bruseo, C, France D.S. Oncology Drug

Discovery Applications Using the FMAT® 8100 HTS System.

J Biomol Screen. 2003 Feb;8(1):81-8.

INFO

For more information on:Applied Biosystems 8200 Cellular DetectionSystem enter: 119

Left: Figure 2.

Staurosporine-inducedapoptosis measuredwith fluorescent dye-labelled Annexin Vto illustrate a screeningassay.

A) Bar graph ofapoptosis data from a96-well FMAT® plate of HCT116 cells treated with 3 µMstaurosporine in wells2a, 3e, 6d,9c, 9f, 10aand 11c.

B) 3D graph illustratinghit analysis using the8200 analysis softwareto plot cell count for Population A(Annexin V-AlexaFluor®647 bindingevents).

C) Well detail of 6c and 7c to showpseudocolour imagesand colour gating. In these examples,Annexin V-AlexaFluor®647 (Dye A) is represented by blueimages and CentriRedstain (Dye B) isrepresented by pink images.

A

B

C

Contact

usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]

AcknowledgementsJulia M. Michelotti,Lolita Evangelista,Applied Biosystems,Foster City, USA

Carol Khodier, Sonia Connaughton,Applied Biosystems,Bedford, USA

arge genotyping projects generate a great quantity ofdata, which need to be managed efficiently to allowrapid and accurate derivation of final linkage or

association results. Some considerations involved in this task are the links between subject details and the relevantgenotype data, the performance of quality control and dataconcordance tests, and the capability to archive and exportfinished data files in a suitable format for downstream linkage or association analyses.

Further benefits come from the ability to import genotype data from different analysis platforms and assay technologies,to draw together all available experimental results into one database.

The new BioTrekker Software v1.0 from Applied Biosystemsaddresses the need to provide a complete solution for genotypingdata management. It allows you to connect to GeneMapper®

Software v3.5 and Sequence Detection System Software v2.2Enterprise database to import analysed genotyping data.

» The GeneMapper Software v3.5 provides upstream raw data analysis from the SNPlex™ system, SNaPshot® kit and microsatellite assays, run on the Applied Biosystems 3730, 3730xl, 3100 and 3100-Avant capillary electrophoresis platforms.

» The SDS Enterprise software analyses data from the ABI PRISM® 7900HT Sequence Detection System, running allelic discrimination assays for SNP genotyping including the comprehensive range of Taqman® SNP genotyping assays from Applied Biosystems.

BioTrekker Software v1.0 offers flexibility in installation:

it can be installed into a GeneMapper v3.5 database instanceon an existing dedicated GeneMapper workstation, or run as a stand-alone system with its own Oracle 8.1.7 StandardEdition database. As a stand-alone system, it uses the same computer specification level as the separate workstationfor GeneMapper Software v3.5, which is equivalent to thatsupplied with our current multi-capillary genetic analysisinstruments (BioTrekker software cannot be supported on Capillary Electrophoresis instrument Data Collection orEnterprise workstations).

The genotyping database manager allows all typing results to be collated in one central database and can create and manage consensus genotypes and performconcordance tests. Quality control tests can be carried out on datasets, with the ability to query and view individualgenotype results when necessary. All valid data can be exportedto text files, enabling downstream statistical analysis toestablish linkage or association results.

Finally, all data can be safely archived, and BioTrekkerSoftware’s security features control database access and canassist in meeting 21 CFR 11 compliance. BioTrekker Softwarev1.0 offers the scientist running a genotyping project themeans to take control of all their data management.

L

AcknowledgementsDave Watts,Applied Biosystems, UK

Contact

usWhether you have a question, query or comment on thisarticle, or any of the others in this issue, please get in touch with us at: [email protected]

For more information on:BioTrekker Software v1.0enter: 120

INFO

product news

It allows you to connect to GeneMapper Software v3.5

and SDS v2.2 Enterprise database to import

analysed genotyping data

BiosystemsSolutions31

New

BioTrekker™

Software v1.0Offers a solution to your genotyping data management problems

32BiosystemsSolutions

he NanoMate 100 with ESI Chip™ from AdvionBioSciences (Ithaca, NY, USA) is a fully-automatedchip-based nanoelectrospray system suitable for

our high-end API mass spectrometers. The NanoMatecombines automation and miniaturisation to increase samplethroughput, sensitivity and information content from tracebiological samples.

The NanoMate combines automated sample handling with the technology of the ESI Chip to produce data from hundredsof samples per day. To analyse samples, the NanoMate uses aconductive pipette tip to draw sample from a 96 well-plate.The sample-filled tip aligns with a nozzle inlet on the back ofthe disposable ESI Chip, creating a tight seal. Each pipette tipand nozzle is used only once, providing a unique path into the mass spectrometer and eliminating sample carryover.Following nanoelectrospray and MS analysis, the NanoMatecan return unused sample to the well or pick up a new sampleand begin the process again.

The ESI Chip

The disposable ESI Chip used with the NanoMate 100 is amicroarray of 100 nanoelectrospray nozzles etched in a siliconwafer, to facilitate stable nanoelectrospray at low flow rates(figure 1). Each nozzle is made to exactly the samespecification so that every nanoelectrospray is identical and provides fully reproducible spray. The ESI chip provides a novel, reliable, and reproducible electrospray of fluid samples for MS analysis.

Metabolite Identification

To illustrate the capabilities of the NanoMate for metaboliteidentification, erythromycin was analysed for metaboliteswithout LC separation using the source on a QSTAR® PulsarHybrid LC/MS/MS System in full MS/MS and precursor ion scanning modes. Erythromycin was incubated at 10 µM with pooled human microsomes for 1 hr at 37

oC to generate

metabolites. Sample preparation consisted of addingacetonitrile to the sample (1:1) and centrifuging for 2 minutes at 8,000 rpm. The supernatant was filtered with a 0.2 µm filter. Both LC/MS, using an ion spray source,and NanoMate/MS analysed the same samples for directcomparisons. Samples were desalted using Millipore ZipTips®

prior to infusion analysis on the NanoMate.

T

product news

NanoMate®

For the QSTAR®, API 3000™, API 4000™ & 4000 Q TRAP™ Systems

The NanoMate combines automation and miniaturisation to

increase sample throughput, sensitivity and information

content from trace biological samples

100 System

Figure 1.

The ESI Chip

Figure 2 shows spectra from the NanoMate and LC/MS runs for an incubated sample of erythromycin. Both spectrawere expanded around the mass range of interest to illustrate the parent and metabolites. This figure clearly showsthat the NanoMate and LC/MS data were comparable.Furthermore the infusion analyses offered by the NanoMateprovide the necessary extended analysis time for precursor ionscanning. Figure 3 shows results of precursor ion scanning withthe NanoMate at a step size of 1.0 amu (top) and 0.1 amu(bottom). The infusion analysis permits the user to acquiremany more data points and thus achieve better data quality for precursor ion scanning.

Mapping Protein Phosphorylation Sites

on a QSTAR System

The extended infusion time obtainable with the NanoMateprovides added flexibility for post-translational mappingstudies. In this example, a standard bovine β-casein trypticdigest was analysed using a precursor ion scan of m/z 79 in negative ionisation mode. Once the phosphopeptides were identified, MS/MS in positive ion was performed in order to sequence the peptides. The results showed that allphosphorylation sites of β-casein tryptic digest could bemapped at 50 fmol/mL. Figure 4 shows the MS/MS spectrumobtained from the phosphopeptide parent ion m/z 1031.4.These results demonstrate that the automated chip-basednanoelectrospray platform is a valuable system forphosphorylation analyses due to stable, extended infusiontimes for completing precursor ion scanning followed byMS/MS on the identified phosphorylated peptides.

Instrument Compatibility

Advion has designed brackets to mount the NanoMate 100 to the QSTAR, API 3000, API 4000 and 4000 Q TRAPLC/MS/MS systems. Applied Biosystems and Advion, via theirstrategic alliance, both ensure system compatibility betweenthe NanoMate 100 and these LC/MS/MS platforms.

Conclusion

The Advion NanoMate 100 robotic system further enhancesthe versatility and performance of the Applied BiosystemsLC/MS/MS systems. In combination with the disposable ESI Chip, the NanoMate allows users to take full advantage ofthe capabilities of nanoelectrospray mass spectrometry for lifescience applications, notably by providing extended acquisitiontime for proteomics and metabolite identification applicationswhere greater sample information is required. Key advantagesinclude higher sample throughput, low sample consumption,high sensitivity, reduced analysis cost and the ability to derivemeaningful information from a very small sample.

Figure 4.

Positive ion MS/MSspectrum of m/z 1031.4from 50 fmol/mL of bovineβ-casein tryptic digest. The spectrumunambiguously identifies the site of serine phosphorylation

Figure 3.

Results from precursor ion scanning with theNanoMate. The precursorsof m/z 158 of erythromycinincubate were investigatedusing a step size of 1.0 amuand 0.1 amu.

Figure 2.

NanoMate (top) andLC/MS (bottom):Erythromycin “Full Scan”TOF (Expanded regionshowing metabolites). The mass spectrum fromthe LC/MS data shown is the average of the LCelution window from 2 to 3 minutes

BiosystemsSolutions33

The ESI chip provides a novel, reliable,

and reproducible electrospray of

fluid samples for MS analysis

INFO

Contact

usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]

For more informationplease contact your localApplied Biosystems Sales Engineer.

AcknowledgementsJean-François Alary,MDS-Sciex,Canada

Colleen Van Pelt,Advion BioSciences Inc.,USA

34BiosystemsSolutions

Background

s research moves more towards understanding protein function, studying protein-protein interactions,

and monitoring changes in protein profiles from systematic challenges, there is a need to developquantitative tools. Recently, there has been an increasing desire to simultaneously compare multiple samples in a relative or absolute manner and a need to develop tags thatprovide broad proteome coverage while maintaining important structural information such as post-translationalmodifications. The requirements have led to the developmentof the iTRAQ reagents.

iTRAQ Reagents

Applied Biosystems iTRAQ reagents are a multiplexed set of non-polymeric, isobaric reagents which yield amine-derivatised peptides from a protein digest, that arechromatographically identical and indistinguishable in MS,but produce strong low-mass MS/MS signature ions thatpermit quantitation. This tagging system allows you to extractmore detailed information from samples because importantinformation, such as post-translational modifications, is notlost in the process.

Since all peptides are tagged, proteome coverage is expandedand analysis of multiple peptides per protein improves the confidence in those identified. Protein identification issimplified by improved fragmentation patterns, with no signalsplitting in either the MS or MS/MS modes and thecomplexity of data is not increased by mixing multiple samplestogether. Additionally, the ability to simultaneously analyse upto four different samples enables the comparison of numeroussample states and provides the flexibility to include duplicates or triplicates into experimental design,

giving the statistical relevance needed for quantitativeexperiments. We have incorporated this quantitationtechnology into a simple workflow of labelling the resultingpeptides after parallel protein extraction and routineenzymatic digestion common to any LC/MS experimentalprocedure. The use of these labels therefore permitssimultaneous measurement of relative and/or absolute protein abundance of multiple, samples in a singleLC/MS/MS run.

Key features and benefits

» Expanded proteome coverage by labelling all peptides,

including those with post-translational modifications

(PTMs) to extract more detailed information from

your samples.

» Enhanced low-level analysis as a result of the signal

amplification from the additive fragmentation of

labelled isobaric peptides resulting in greater depth into

sample information

» Increased confidence in identification and quantitation by

tagging multiple peptides per protein to gain more

statistically significant information.

» Analysis of up to four different biological samples

simultaneously in a single experiment.

» Perform absolute quantitation across numerous sample

states, for the synchronous uniform comparison of normal,

diseased and/or drug treated states.

» Simple workflow labels peptides allowing rapid progression

to LC/MS/MS analysis and easy data interpretation

with Pro QUANT Software for relative and absolute

quantitation.

INFO

For more information on:iTRAQ Reagentsenter: 121or contact your localApplied Biosystems Sales Engineer

AcknowledgementsLynn Zieske, Applied Biosystems,Foster City, USA.

Tony Hunt, Applied Biosystems,Framingham, USA

Expanded proteome coverage by labelling all peptides, including

those with post-translational modifications (PTMs) to extract

more detailed information from your samples

product news

New Amine SpecificiTRAQ™ Reagentsexpand multiplexing and quantitationcapabilities for Proteomic researchers

A

Analysis of up to four different biological

samples simultaneously in a

single experiment

BiosystemsSolutions35

The iTRAQ Reagent structure

Comprises of two main segments:

1. Peptide Reactive Group (PRG) – Covalently links the iTRAQ reagent to the free primary amines (N-terminal amines and lysine side chains) in the peptide.

2. Isobaric Tag – Isobaric, by definition, implies that any two or more species have the same atomic mass but different arrangements. Choosing an isobaric tagging system enables multiple samples to be tagged with no resulting increase in MS complexity when quantitation occurs upon fragmentation in MS/MS space. As illustrated in figure 1, the iTRAQ reagent tag consists of reporter group (tag after MS/MS fragmentation) and balance group (to keep the overall isobaric tag mass the same for each reagent.The balance portion of this reagent is ultimately invisible upon fragmentation at the MS/MS Fragmentation Site. During MS/MS, fragmentation occurs on both sides of the balance group, resulting in neutral loss of the balance group and releasing the reporter group. The reporter groups appear in the low mass region between m/z 113-119, an area specifically selected because no common fragment ions have been found to appear in that region.

Isobaric tagging of peptides for multiplex analysis

A key feature of the iTRAQ reagents is, when combined, the MS of the iTRAQ Reagent-labelled sample digest mixture resembles the MS of an individual sample (assumingthe same peptides are present). The balance group ensures that an iTRAQ reagent-labelled peptide has the same m/z,whether labelled with iTRAQ Reagent 114, 115, 116, or 117.In the example illustrated, a six protein mix was labelled5:1:10:1 with iTRAQ reagents 114, 115, 116 and 117,respectively. The samples were mixed and the resulting MS and MS/MS spectra recorded using a QSTAR® XL system. As seen, there is no increase in complexity in the overall spectra.There is a single parent ion in the MS spectrum (see figure 2inset), cumulative from all four samples. In addition, the y- andb- ion rich MS/MS spectra is also not complicated by multiplesignals except in the important reporter region in which therelative ratios of the three species are recorded.

Absolute quantitation

Quantitation of specific proteins of interest can also beperformed using the iTRAQ reagents. This involves comparing peptides of interest of the targeted protein(s) to known (quantified) amounts of labelled synthetic standardpeptides, representing unique fragments of the protein(s) of interest, spiked into the sample. By labelling the spiked in peptide with one of the iTRAQ reagents and the sample(s) labelled with one or more of the other reagents, both relative and absolute quantitation can be obtained in asingle MS/MS spectrum.

Software for data analysis

The Pro QUANT software package supporting the iTRAQReagents is available for the QSTAR® XL and Q TRAP® familyof MS systems.

Conclusions

Whether you are performing protein expression analysis or absolute quantitation experiments, Applied BiosystemsiTRAQ reagents coupled with our MS instrumentationprovide the features, the confidence, and the statisticalrelevance you need for quantitative biology. In addition to PTM analysis, these reagents are also ideal for laboratories performing discovery/validation analyses and time course studies.

Contact

usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]

= Fragmentation site

Above. Figure 1.

VLVDTDYK

Above. Figure 2.

QSTAR XL system

Q TRAP system

36BiosystemsSolutions

product news

or most high-throughput biological laboratories, anincrease in throughput means an increase in operatingcomplexity. Managing an ever more complicatedworkflow has become vital for a laboratory to increase

productivity, improve data quality, reduce costs, and integrate datafrom many different sources.

Recently released, the Applied Biosystems LIMS is a revolutionary new workflow management, process automation, and data integration solution for the high-throughput biological laboratory.

Applied Biosystems LIMS includes a comprehensive set of featuresto help you integrate, automate, and manage all your data andworkflow processes. The software is built on the fully scalable,extensible, multi-tiered architecture from Applied Biosystems andleverages the core sample management functionality found in theiraward-winning SQL*LIMS® Software. This software is theLaboratory Information Management System (LIMS) of choice for more than 30,000 professionals in 1,000 laboratories across awide range of industries.

The features of Applied Biosystems LIMS allow you to:

Manage samples and containers and organise data» Map sample transfer and re-array from container(s) to

container(s) using a flexible container mapping tool

» Track samples and containers through their life cycles

» Organise and track experiment and study data with user-defined project folders

F

a revolutionary new workflow management, process automation,

and data integration solution for the high-throughput

biological laboratory

Applied Biosystems LIMS

for the Life Sciences

INFO

For more informationVisit:www.appliedbiosystems.com/InformDisc or email:[email protected]

Related articles can be found on:Pages 21, 26 & 50

AcknowledgementsMaria Rodrigues,Lori Graham, Applied Biosystems,USA

Transforms the way your laboratory operates

Support a changing laboratory operation

with flexible, adaptable features » Easy-to-use, drag-and-drop, graphical workflow editor

» Reusable activities, protocols and workflows

» True workflow engine for automatic job queue and data flow

» Fully documented API and developer tool kit

» Library of specific Applied Biosystems and third-party instrument interfaces

» Result parser for importing results from third-party instruments

» Out-of-the-box workflow to support Applied Biosystems Taqman® Gene Expression, SNP Genotyping Assays and SNPlex system

» Pluggable user interface in activity-driven laboratory console

Maximise laboratory process and research efficiency» Track consumables and sample inventory

» Monitor status of ongoing laboratory operations and instruments using the process viewer

» Promptly notify users of process errors using user-defined notification feature

» Manage assays by markers and track assay reagents

Support data security and assist with

21 CFR Part 11 compliance» Control access by data group and jobtype functions with

user-configurable feature

» Get user-configurable audit trail and electronic signature

Applied Biosystems LIMS is truly the next generation laboratorymanagement and automation software. It is designed to transformthe way your laboratory operates, helping you integrate, automate and manage all your laboratory data and workflowprocesses. Informatics systems from Applied Biosystems are backed by our global support, professional services, and Informatics Specialists worldwide.

BiosystemsSolutions37

Figure 2.

Activity-driven Laboratory Console to view and manage daily laboratory tasks and work lists.

Figure 1.

Drag-and-dropgraphical workflow

editor to define end-to-endlaboratory

workflow process

38BiosystemsSolutions

product news

The Applied Biosystems 7300 Real-Time PCR System is theperfect entry to real-time PCR, producing high-end resultswithin budget. The System uses a standard 96-well format.The filter wheel with four emission filters allows the detectionof all dyes between 510nm and 650nm. Through an advancedmulticolour detection algorithm the system can analyse up to4 dyes simultaneously and enables you to perform a widevariety of applications – including gene expression analysis,pathogen quantification, SNP genotyping and +/- assays toconfirm the presence or absence of specific targets.

For all customers who need to use a broader range offluorophores, the Applied Biosystems 7500 Real-Time PCRSystem is the right choice. The system has an advanced opticunit with two 5-filter wheels, which give a higher sensitivityespecially for redder dyes. The Relative Quantitation (RQ)Study software is a standard component of the 7500 system,while it is optional for the 7300 instrument. The RQ Studysoftware allows you to generate gene expression profiles fromup to ten 96-well plates in seconds. The really revolutionarycharacteristic, however, is the new high-speed thermal cyclingupgrade option. All users who need real-time PCR resultsquickly can obtain them in less than 40 minutes, in a singletube format or using a 96-well plate. The 7500 system offersadditional performance across the same range of applicationsas the 7300 system.

The ABI PRISM® 7900HT Sequence Detection Systemcompletes the series of real-time PCR instruments fromApplied Biosystems. The system offers unmatched flexibilityand throughput with user interchangeable thermal cyclingblocks and an optional Automation Accessory for automaticplate loading and unloading for true walkaway automation.Currently 384-well, 96-well or TaqMan® Low Density Arraythermal cycling blocks are available. The TaqMan Low DensityArray is a custom-configured 384-well low volume microfluidic card (2µL reaction volume per well) which allows low-density gene expression array experiments to be performedwithout the need for sample handling robotics. TaqMan® GeneExpression Assays are pre-deposited and dried down into thereaction wells during the manufacturing process, leaving onlya simple sample loading process to be performed by the systemuser. The result is an extremely easy-to-use low density array with the gold standard data quality provided by Applied Biosystems TaqMan Gene Expression Assays and real-time PCR systems.

The TaqMan Low Density Array can accommodate between12 and 380 gene targets, up to 8 samples per array, and offersthree choices of replicate numbers. The 7900HT system alsooffers a comprehensive software suite which simplifies high-throughput data analysis and provides a comprehensiveset of features for users working in a validated environment.

The ABI PRISM 7900HT Sequence Detection System isApplied Biosystems flagship real-time PCR system, and is the most capable, most flexible and highest throughput system available from Applied Biosystems.

With the introduction of the Applied Biosystems 7300 and 7500

Real-Time PCR Systems, we can now offer three different

instruments for all possible requirements and applications

INFO

For more information on:Applied Biosystems 7300Real-Time PCR Systementer: 122

For more information on:Applied Biosystems 7500Real-Time PCR Systementer: 123

For more information on:ABI PRISM 7900HTSequence DetectionSystem enter: 124

AcknowledgementFalko Kraeusche,Applied Biosystems,Germany

Real-TimePCR platforms from Applied Biosystems

– instruments to suit all requirements ince Applied Biosystems pioneered real-time PCR in 1995, we have continued to develop the

technology to provide more powerful solutions for labs of all sizes. With the introduction of the

Applied Biosystems 7300 and 7500 Real-Time PCR Systems, we can now offer three different

instruments for all possible requirements and applications.

Which instrument is best suited to your requirements?

S

Announcingthe family of

BiosystemsSolutions39

Applied Biosystems 7300Real-Time PCR System

Fixed 96-well

No

Halogen lamp, fixed wavelength

4 colour detection

No

No

Absolute quantitation (standard curves),Allelic Discrimination (SNP genotyping)and Plus/Minus assays using anInternal Positive Control

Yes

Yes

Relative Quantitation Study(comparative CT method)

No

Yes

Full-featured instrument that provideshigh-end results within budget

Depending on your application, throughput needs and budget you can now choose between three real-time PCRinstruments from Applied Biosystems. In an integrated scienceapproach, the real-time PCR instruments combined with our high-quality sample preparation, optimised real-time PCR chemistry, validated TaqMan Gene Expression Assays orCustom TaqMan Gene Expression Assays, these instruments offeryou a complete solution for your Real-Time PCR experiments.This gives you reliable results easier and faster.

Researchers in genomics can speed up their projects still further by taking advantage of rich genomic information provided in the myScience research environment:http://myscience.appliedbiosystems.comthe PANTHER™ Classification System for molecular functionand biological processes, or the subscription-based CeleraDiscovery System™ Online Platform.

Block Format

Support forTaqMan LowDensity Array

Excitation Source

Emission

Future upgrade tofast thermal cycling

Automated PlateLoading/Unloading

SoftwareApplications/AssayTypes

Automatic CT

determined

Automated AlleleCalling

Optional Software

Software featuresand functions tosupport customersrequiring validation

Includes PrimerExpress™ Software

Summary

Applied Biosystems 7500Real-Time PCR System

Fixed 96-well

No

Halogen lamp, variable wavelength

5 colour detection

Yes

No

Absolute quantitation (standard curves),Relative Quantitation Study(comparative CT method), AllelicDiscrimination (SNP genotyping) andPlus/Minus assays using an InternalPositive Control

Yes

Yes

No

Yes

Additional performance and features inthe 96-well format

ABI PRISM 7900HTSequence Detection System

User interchangeable 96-well, 384-welland TaqMan Low Density Array*

Yes

Laser

Continuous wavelength detection from 500-660nm allows highly flexiblemulti-colour detection capabilities

Yes

Yes, with optional Automation Accessory

Absolute quantitation (standard curves),Relative Quantitation Study (comparativeCT method), Allelic Discrimination (SNP genotyping)

Yes

Yes

Enterprise database software to supporthigh-throughput gene expression andSNP data collection and analysis

Yes, software includes functions andfeatures to support customers requiring21CFR part 11 compliance

Yes

Most capable, flexible and high-throughput real-time PCR instrument

* TaqMan Low Density Array requires an initial service upgrade, after which the thermal cycling block is user interchangeable

40BiosystemsSolutions

product news

This new version is the first fully web enabled LIMS and is a complete enterprise-accessible solution for pharmaceutical and

other QA/QC manufacturing operations. This new system’s extended power simplifies large, multi-site deployments;

reduces personnel overhead costs; and provides new, easy-to-use, streamlined navigation

pplied Biosystems announces SQL*LIMS v5software, an enhanced version of this award-winning Laboratory Information Management

Systems (LIMS). This new version is the first fully webenabled LIMS and is a complete enterprise-accessiblesolution for pharmaceutical and other QA/QCmanufacturing operations. This new system’s extendedpower simplifies large, multi-site deployments; reduces personnel overhead costs; and provides new, easy-to-use, streamlined navigation.

First Fully Web-enabled LIMS

SQL*LIMS v5 software’s new, open Web services platform has been built using industry standard technology including the Oracle® Application Server 10g and Database 9i, and supports the extensible Java 2 Platform,Enterprise Edition (J2EE) standards-based application. This powerful platform allows you to build and extendfunctionality to meet your complete needs for e-commerceand web messaging.

Further, typical LIMS applications require you to installsoftware on local user workstations, making the systems morecostly to implement and maintain. With the introduction ofthe Web-enabled SQL*LIMS v5, you can reduce your costswith a more lightweight, configurable system that is easy todistribute, maintain, and connect to other critical applications.You also get full Web access from inside or outside yourfirewall. And you can do all this while still securely protectingyour data quality and integrity.

Reduce Your Costs

A comprehensive LIMS is critical for today’s QA/QCmanufacturing organisations to maximise automation,optimise laboratory processes, and create a truly paperlesslaboratory. Consequently, the return on investment of a LIMS is usually significant, with a quality system reducingstaff errors, identifying process bottlenecks, increasing analysis accuracy, and reducing material and reagent waste. A LIMS can also potentially reduce your project turnaround time from years to months.

New Expanded Features

Applied Biosystems greatly extends the power of earlier releaseswith major new features and functionality in SQL*LIMS v5. The new software can be easily configured to meets both therequirements of a small laboratory or large, global operation.

New expanded features include:

» Instrument calibration management

» Analyst training and qualification tracking

» Reagent inventory tracking

» Streamlined, two-mouse click navigation

» Enhanced fill-down, pop-ups for data entry and approvals

» Secure single user password for users of multiple

database instances

A

SQL*LIMS®

v5 SoftwareThe best in LIMS just got better

INFO

For more informationvisit: www.sqllims.com

Or email:[email protected]

AcknowledgementsLori Graham, Vince Woodall, Applied Biosystems, USA

Peter Boogaard, Applied Biosystems, The Netherlands

BiosystemsSolutions41

Proven Quality and Performance, Fully Validated

SQL*LIMS Software is the industry’s most innovative, end-to-endsolution to integrate, automate, and manage laboratory data andworkflow processes. That is why it is the LIMS of choice for more than 30,000 professionals in 1,000 laboratories across a widerange of industries.

SQL*LIMS v5 Software is “customer validation ready,” allowing you to easily tailor the standard application to meet your needs. Internal teams audit the Applied Biosystems Quality Program regularly and independent auditors from UL and PDA/ARC certify and document Applied Biosystemsprocesses for customers in regulated industries.* An audit is on file in the PDA/ARC Repository.

All informatics systems from Applied Biosystems are backed by our global support, professional services, and informaticsspecialists worldwide. *Underwriters Laboratories, Inc. (UL) is a leader in U.S. product safety

and certification worldwide. PDA is a non-profit international association of scientists involved in developing, manufacturing, and regulatingpharmaceuticals/ biopharmaceuticals and related products. PDA developedthe standardised audit process used by suppliers of computer products andservices for regulated pharmaceutical operations. The Audit RepositoryCenter (ARC) serves as the global repository for all audits.

Figure 1.

SQL*LIMS v5 software includes expanded functionality for instrument calibration, user qualification and reagent inventory tracking

SQL*LIMS Software is the LIMS of choice for more than 30,000

professionals in 1,000 laboratories across

a wide range of industries

Figure 2.

New, streamlined navigation makes SQL*LIMS v5 software easier to learn and use.

Contact

usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]

42BiosystemsSolutions

product news

Measuring gene expression in whole blood is becoming an increasingly important research tool. ‘Global’ measurement

of expression changes in whole blood by microarray technologies permits the profiling of

many thousands of mRNA transcripts simultaneously in a single sample.

IntroducingTempus™ Blood RNA Tubes

INFO

For more information on:Tempus Blood RNATubes enter: 125

Or visit:http://info.appliedbiosystems.com/tempus

For more information on:ABI PRISM 6100 NucleicAcid PrepStationenter: 126

Stabilise RNA profiles in whole blood for 5 days!he Tempus Blood RNA Collection Tube isrevolutionising gene expression measurementby significantly extending your RNA profiling

capability. When combined with the ABI PRISM™ 6100Nucleic Acid PrepStation, this unique solution delivers a host of benefits:

» Stable RNA and transcript profiles in 3 mL of whole blood for up to 5 days at room temperature

» Unequalled real-time PCR and microarray analysis results

» Fast, easy purification of total RNA from up to six Tempus tubes simultaneously in 45 minutes

» Superior RNA quality

Measuring gene expression in whole blood is becoming an

increasingly important research tool. ‘Global’ measurement of expression changes in whole blood by microarray

technologies permits the profiling of many thousands of mRNA transcripts simultaneously in a single sample.

Real-time PCR assays based on TaqMan® probes or

SYBR® Green 1 Dye can be performed on a smaller number

of mRNA transcripts (also known as biomarkers), in a largerpopulation group to understand the importance of these

expression changes. This can be of significant help in gaining

an in-depth molecular profile of a disease, or understanding the impact of various treatments.

Isolation of high-quality RNA from whole

blood samples is difficult

Recent work at Applied Biosystems1 has shown that the

task of isolating high-quality RNA is complicated further

because under ambient conditions, expression profiles areunstable on a timescale of less than one hour.

This instability is the result of metabolic activity that continues

in standard blood collection tubes post-draw (see figure 1).

Up-regulation of ~50-fold has been measured for some gene

targets after one hour at room temperature. Because blood is

often drawn at sites remote from the investigating team,

there is often a time lag (typically hours but sometimes days),

before the RNA isolation process can begin. During this time

lag, ex vivo changes in expression profiles can occur.

For accurate measurement of gene expression profiles,

transcription must be stopped at the time of the blood draw.

The Tempus Blood Tube is designed for direct isolation of

3 mL of patient blood into a standard plastic, evacuated blood

collection tube containing the Applied Biosystems RNA

Stabilising Reagent. The RNA, and therefore the expression or transcript profile, is stable in the collection tube for

up to five days at room temperature (18–25°C), or longer at4°C (figure 2). The stabilised blood may also be frozen in

the collection tube for prolonged storage or transport.

T

Figure 1.

Gene expression profile in non-stabilised whole blood (Tanner et al., Clin. Lab. Haem. April 2002, 24,337–341)

BiosystemsSolutions43

A simple 45-minute protocol on the ABI PRISM 6100 Nucleic AcidPrepStation, purifies the RNA and generates high-quality nucleicacid (A260/280 > 1.9) at yields of 2–8 µg/mL of human wholeblood. Up to six tubes can be processed simultaneously, producingtotal RNA that is free of RT/PCR inhibitors and other enzymes. The RNA is suitable for all downstream applications, includingreal-time PCR and microarray analysis.

Safe, high-quality VACUETTE® tubes

The Tempus Blood RNA Tube was developed collaboratively by Applied Biosystems and Greiner Bio-One GmbH, one of the world’s leading suppliers of blood collection products wellknown for both quality and safety. The tubes were the first to be manufactured from the virtually unbreakablepolyethylene terephthalate (PET) plastic and can withstandfreeze/thaw procedures.

RNA isolation Chemistry

Applied Biosystems has developed a unique RNA extractionmechanism that generates the highest quality total RNA andrequires only a small number of steps.

Applied Biosystems RNA chemistry isolates a representative andproportional selection of messenger, ribosomal and small nuclearRNAs. It does not isolate tRNA, mammalian 5S ribosomal RNA,highly degraded RNA, or any RNA species less than approximately200 nucleotides. This, along with very low protein contaminationof the RNA, provides distinct advantages over other extractionchemistries, in which tRNA and 5S ribosomal RNA oftencomprise 15–20% of total RNA.

References

1. Tanner et al., Clin. Lab. Haem. 2002, 24, 337–341

The tubes were the first to be manufactured from the virtually

unbreakable polyethylene terephthalate (PET) plastic

and can withstand freeze/thaw procedures

To find out more about high performance,

low-cost prep solutions, visit:

http://info.appliedbiosystems.com/prep01

Figure 2.

Comparison of gene expression stability from blood stabilised in Tempus Blood RNA Tubes and standard EDTA blood collection tubes.

Applied Biosystems has developed a unique RNA extraction

mechanism that generates the highest quality total

RNA and requires only a small number of steps

44BiosystemsSolutions

promot ions

*Terms and conditions for this special offer: Orders can be placed by email, fax or phone quoting a purchase order number and cycler offer. Offer valid until 31 July 2004.

No other discounts apply. All prices exclude delivery and local tax. E&OE. Available for Europe only.

The GeneAmp® PCR System 9700 thermal cycler is a high-performancethermal cycler with built-in flexibility, provided by different blocktemperature modes and a range of user-interchangeable PCR block options.

» User-friendly graphical display for intuitive programming

» Excellent temperature uniformity and accuracy for reproducible results

» Networking software for central control of up to 31 cyclers available

The GeneAmp® PCR System 2700 thermal cycler has been designed to meetthe needs of researchers who need to amplify nucleic acids, and rely upon adedicated, basic thermal cycler for PCR or cycle sequencing.

» Fixed sample block, 96-well format

» Easy-to-use graphical user interface

» Compact design maximises bench space

As an owner of a GeneAmp® PCR System TC1, 480, 2400, or 9600,until 31 July 2004, you're eligible for a 25% discount when you moveup to a GeneAmp® PCR System 2700 or 9700 thermal cycler

Thermal Cycler Part No. Special Offer

GeneAmp PCR System 2700 4322620 25% discount

GeneAmp PCR System 9700 with 96-well aluminum block 4314879 25% discount

GeneAmp PCR System 9700 with 60-well 0.5ml block 4310899 25% discount

GeneAmp PCR System 9700 with 96-well gold block 4314878 25% discount

Optimise your PCR performance levels

SpecialSpecial

SpecialSpecialGeneAmp® PCR Systems2700 and 9700 Thermal Cyclers

PLEASE NOTE: The additional 10% discount is currently only available to those countries listed above. # For Italy the offer is only valid for payments within 30 days.*Terms and conditions for Gold Offer: Orders can be placed by email, fax or phone quoting a purchase order number and Gold Offer. Special Offers valid until 31 July 2004. No other discounts apply. All prices exclude delivery and local tax. E&OE. Available for Europe only. **Terms and conditions for Gold Online Offer: Orders can be placed only throughour online store. Special Offers valid until 31 July 2004. No other discounts apply. All prices exclude delivery and local tax. E&OE. Available for Europe only.

BiosystemsSolutions45

OffersOffersOffers

Gold Offer Gold Online OfferUntil 31 July 2004 - save 45% Until 31 July 2004 - save 55%

The Enzyme of choice for most PCRApplications... AmpliTaq Gold® DNA Polymerase

AmpliTaq Gold® DNA Polymerase with Gold Buffer:

6 x 250 units or 12 x 250 units AmpliTaq Gold DNA Polymerase withGeneAmp® 10X PCR Gold Buffer and MgCl2 at 45% discount from list price

Package Part No. Special Offer6 x 250 Units 4311814 45% discount12 x 250 Units 4311820 45% discount

AmpliTaq Gold DNA Polymerase with Buffer I:

6 x 250 units or 12 x 250 units AmpliTaq Gold DNA Polymerase with GeneAmp 10X PCR Buffer I at 45% discount from list price

Package Part No. Special Offer6 x 250 Units N808-0242 45% discount12 x 250 Units N808-0244 45% discount

AmpliTaq Gold DNA Polymerase with Buffer II:

6 x 250 units or 12 x 250 units AmpliTaq Gold DNA Polymerase with GeneAmp 10X PCR Buffer II and MgCl2 at 45% discount from list price

Package Part No. Special Offer6 x 250 Units N808-0243 45% discount12 x 250 Units N808-0245 45% discount

Austria20236109

Belgium 20235930

Denmark 20235939

Finland20235933

France 20235934

Germany 20235931

Ireland 20235935

Italy #

20235936

Netherlands20235937

Norway20235941

Portugal 20235938

Spain 20235932

Sweden 20235942

Switzerland 20236108

United Kingdom 20235940

Save 55% by ordering the special offer packages above throughour online store – https://store.appliedbiosystems.com – this gives you an EXTRA 10% discount.

When ordering please refer to Special Offer.

Promotional codes for online ordering

46BiosystemsSolutions

he ABI 433A Peptide Synthesis System, equippedwith feedback control based on UV Monitoring,

allows you to create even longer and more complexpeptides efficiently.

Synthesis will be more specific and sensitive and synthesistimes can be shortened.

T

Part No. Description Reference Code Discount from List Price

4317033 HATU Kit HATUOF1 30%(19g HATU and DMF)

GEN076521 HATU, 5G HATUOF2 30%

GEN076523 HATU, 25G HATUOF3 30%

GEN076525 HATU, 100G HATUOF4 30%

401571 8 ml Reaction Vessel PEPSYN1 50%for the 433A

401573 41 ml Reaction Vessel PEPSYN2 50%for the 433A

4335867 Real-Time UV Monitoring PEPSYN3 50%Accessory for the 433A

4344180 SynthAssist v3.0 Software PEPSYN4 50%Upgrade Kit incl. computer

Figure 1.

Improved synthesis of ‘difficult peptides’

You can take advantage of our special promotional offer*

and try out the most versatile activator chemistry,

HATU, or upgrade your 433A Peptide Synthesis System

with UV Monitoring capability or upgrade to the latest

PC based Software SynthAssist v3.0.

For ordering, please use the part number and refer to the respective reference code below.

Achievebetter, faster synthesisand superior peptides

*Terms and conditions for this special offer: Orders can be placedby email, fax or phone quoting a purchase order number. Offer validuntil 31 October 2004, for confirmed orders. No other discountsapply. All prices exclude delivery and local tax. E&OE.

Until 31 October 2004

promot ions

BiosystemsSolutions47

erman researcher Professor Dr Michael Przybylski

is the latest scientist to receive a coveted award

sponsored by Applied Biosystems, which recognises

individuals who have made a significant contribution to the

development and application of mass spectrometry in life

sciences. Professor Przybylski, who is head of the Laboratory

of Analytical Chemistry at the University of Konstanz,

was selected by a scientific jury in recognition of his

outstanding work in the area of protein analysis.

The jury was appointed by the Board of the German

Society of Mass Spectrometry and led by Professor Dr. Jasna

Peter-Katalinic, who acknowledged his work in her award

speech: “In the past two decades, mass spectrometry has

undergone important developments and its use in genomics and

proteomics has effectively revolutionised life science research.

Novel concepts have allowed mass spectrometry to be used in a

more dynamic way and Professor Przybylski has contributed to

these developments from a very early stage.

In the eighties, he used plasma desorption mass spectrometry

for the analysis of intact biopolymers and, in more recent years,

has been working on Fourier transform ion cyclotron

resonance (FTICR) mass spectrometry. The results of this

work have given extraordinary insight into the biologically

active conformations of proteins and their pathophysiological

changes, for example, in the course of Alzheimer’s disease,

where it has recently provided the elucidation of a new

vaccine lead structure (Nature Medicine, 2002)”.

More than 250 original research articles published

Professor Przybylski’s research is currently focusing on

proteomics of neurodegenerative proteins, applications of

mass spectrometry in immunology and the identification

of the chemical structures of molecular recognition by

antibodies. He has published more than 250 original research

articles, given approximately 100 invited lectures and is

co-editor of several scientific journals. His research is involved

in numerous international collaborations with European

laboratories and with the National Institutes of Health in the

US. He is adjunct guest professor of the Chinese and the

Hungarian Academy of Sciences, and has received an honorary

doctoral degree from the A.I. Cuza University of Iasi (Romania).

The award and a prize of 5,000 Euros was presented by

Professor Peter-Katalinic in a ceremony held at the UFZ

Centre for Environmental Research, Leipzig-Halle GmbH at

the Helmholtz Association, in Germany.

Applied Biosystems expresses its warmest congratulations toProfessor Przybylski and wishes him every success in the future.

G

“In the past two decades, mass spectrometry has undergone

important developments and its use in genomics and proteomics

has effectively revolutionised life science research”

Above.

left to right:

Professor Dr Juergen Grotemeyer,

University of Kiel;

Professor Dr Jasna Peter-Katalinic,University of Muenster;

Professor Dr. Michael Przybylski,University of Konstanz,

Dr Holm Sommer,Applied Biosystems

German researcher wins Applied Biosystems awardfor excellence in mass spectrometry

customer focus

ocumentation of proper installation of laboratoryequipment, and its intended use, is a basicrequirement in a regulated environment. Good Laboratory Practices (GLP), Good

Manufacturing Practices (GMP), Good Clinical Practices (GCP),ISO 9000 and other international standards define the needs for an operation which at the end delivers the traceablerecords and documents during the lifecycle of an instrument.

The ordered instrument has to be appropriate for the application it is needed for. This is part of the Design Qualification (DQ). The specification sheet is a valuable source of information about the guaranteed characteristics of the system. During the sales process a thorough review of the needed features compared with the offered features, together with the Sales representative, will lead into the purchase of the right system.

At the point of installation an Installation Qualification (IQ) is required. The IQ assists in documenting what had been previously ordered and also been delivered. Is it the right model?What about the purchased accessories? Is the software complete? At what firmware is the system running? Which serial numbers need to be recorded? After this has been noted down into theprotocol, the instrument will be installed according to installation requirements.

An upfront delivered pre-installation guide in combination with the requirements discussed during the sales process will allow planning of the laboratory environmental operatingconditions. To have those conditions right is a prerequisite for a successful installation. A check-up of the instruments basic functions is the end of the IQ part.

The Operational Qualification (OQ) or Instrument PerformanceVerification (IPV) will follow immediately after the IQ. Now theverification of the offered specifications, according to thespecification sheet of the instrument, proves that the instrument isworking as expected. Those steps are either outlined in detail directlyin the protocol or there is a reference taken to other supportingdocuments, which are provided in the IQ/OQ package. Collateralrecords and reports are added to the OQ protocol where applicable.

The installation done by a Field Service Engineer (FSE) includes theset-up of the instrument, the verification of the installationspecification as outlined by Applied Biosystems and also theintroduction into the basic operation of the instrument. Those whohave fully attended the training session during the installation arerecorded in the Logbook as trained users.

The IQ/OQ protocols are always product specific and as the IQ/OQis very much tight into the on-site processes defined within thelaboratories SOP’s, the documentation is provided to our customersas soon as the purchase is done. This gives the time to allow thereview of the protocol and to adopt the necessary changes to theSOP’s in order to link all required documents smoothly into eachother. A pre-approval of the document by the quality personnel atthe customers’ site and the person executing the steps in the protocolwill document that this has happened.

As adequate maintenance of the system is demanded by the FDA,other services are offered by Applied Biosystems such as ServiceAgreements including Planned Maintenance (PM). To combine forinstance an annual PM with an annual OQ of the instrument is aliked combination by those customers who have incorporated ourrecommendation into their SOP’s.

A subject that is often discussed between the quality department atthe customers’ site and the manufacturer is the question whether theOQ has to be performed after a repair. To help in this discussion wehave worked out a list of spare parts for each instrument, which arecrucial for the instruments specifications.

D

48BiosystemsSolutions

Documentation of proper installation of laboratory

equipment, and its intended use, is a basic

requirement in a regulated environment

customer focus

Supporting the Qualification process in your laboratorywith the appropriate documentation and agreements

BioQualificationSM

Service

BiosystemsSolutions49

This spare parts list is available in the protocol for the customersto judge to what extent an OQ has to be re-done in the eventof a repair, where one of those spare parts has been exchanged.

For all instruments installed by a FSE, the execution of theIQ/OQ is part of the BioQualification package price. The FSEis fully trained on the instrument and his/her trainingcertificates are available on request. Part of our qualityphilosophy is to have always best educated and knowledgeableengineers employed, this also requires training on the IQ/OQexecution and a constant update on all changes happening tothe system.

The tools to align and adjust the instrument under PM or service are carried by all FSE’s. Those tools are manufacturedby Applied Biosystems or available as standard tools on themarket. When needed both sorts of tools are calibratedaccording to their calibration cycle. All tools that requirecalibration are tracked in our system and are traceable tointernational standards.

BioQualification products are available for a broad range of instruments we offer (e.g. Real-Time PCR Systems, Mass Spectrometers, Genetic Analysis Products and others),and can be combined with the various Service Agreements such as BioAssuranceSM Plus, BioMaintenanceSM, etc.

Part of our quality philosophy is to always have

the best educated and knowledgeable

engineers employed

For more information:Contact your local Applied Biosystems office,Sales Representative or Field Service Engineer

AcknowledgementRobert Keidl, Applied Biosystems, Germany

INFO

50BiosystemsSolutions

customer focus

High throughput

Genotypingusing ABI PRISM® 7900HT

Sequence Detection System

This project will include approximately 70 genes and up to 350 SNPs. The combined size of

cohorts will be up to 2,000 individuals for each study. Thus, this project

will require completion of 700,000 genotypes

pplied Biosystems, Genoscreen (Lille-France) and theGenetics of Multifactorial Diseases Laboratory (Lille-France) initiated a collaborative project in

September 2003 using high-throughput genotyping to discover new susceptibility genes for type 2 diabetes and obesity. This project will include approximately 70 genes and up to 350 SNPs. The combined size of cohorts will be up to 2,000individuals for each study. Thus, this project will requirecompletion of 700,000 genotypes.

Type 2 diabetes and obesity candidate genes were

selected based on several criteria including:

» chromosomal localisation near an obesity/type 2 diabetes–linked locus in human or animal models

» expression profile in response to modification of the environment in a variety of tissues (including adipocytes, hypothalamic neurons, pancreatic beta cells or skeletal muscle)

» role in physiological pathways such as food intake, insulin-resistance and insulin secretion

» targeted gene disruption or transgenic over-expression modifying phenotypes associated with obesity and/or type 2 diabetes in animal models.

A

A partnership between a

CNRS laboratory and the

Applied Biosystems and

Genoscreen companies

ABI PRISM 7900HT Sequence Detection System

BiosystemsSolutions51

The “common disease, common variant” hypothesis formultifactorial diseases (Reich and Lander 2001), implies that typing of the most frequent SNP representative of each candidategene haplotype block (covering up to 20-30 kb or more) shouldallow enough statistical power to detect linkage disequilibrium(LD) between a functional SNP located in this block and thecomplex trait under study. The association can be detected even ifthe genotyped SNP is not itself functional but rather is in LD with the causative variant(s).

The most informative, high frequency SNPs, regularly spacedthroughout the entire gene sequence from the 5‘ region to the 3‘ region of each candidate gene, were selected using theSNPbrowser™ data visualisation tool. The selected SNPs aregenotyped in the case and control DNA samples and the genotypeand haplotype SNP frequencies are compared in subjects withfamilial T2D versus non-diabetic subjects and also between obeseversus non-obese subjects. In addition, for each SNP, analysis ofvariance of intermediate quantitative traits tests will be performed.More than 100 obesity and type 2 diabetes related phenotypes areavailable including BMI, leptin, insulin secretion and insulinsensitivity indexes, lipids, insulin and glucose during oral glucosetolerance test (OGTT).

In addition to bringing considerable amounts of novel geneticinformation to the field of metabolic disease research, this studydemonstrates the viability and the efficiency of the ABI PRISM®

7900HT Sequence Detection System (TaqMan® assay technologyfrom Applied Biosystems) for high throughput genotyping (up to 30,000 genotypes a day) and the technical advantages thatgenomic platforms like Genoscreen can provide to researchers.

The combination of SNP selection strategy (SNPbrowser software), SNP genotyping technology, a high throughput SNP genotyping platform (Genoscreen), biostatistic resources(Laboratory of multifactorial diseases) described here cancontribute to a powerful and rapid identification of potentialsusceptibility genes for multifactorial diseases. Furthermore, this approach can save time and manual labour.

Additional genetic and functional studies will be required toidentify the ‘disease causing’ SNPs that are associated with thedevelopment of T2DM and obesity.

Figure 1.

Identification of susceptibility genes for Obesity and type 2 Diabetes.

The association can be detected even if the genotyped

SNP is not itself functional but rather is in

LD with the causative variant(s)

Figure 2.

For more information on:ABI PRISM 7900HT Sequence Detection System enter 127

To download SNPbrowser Software visit:www.appliedbiosystems.eu.com/mk/get/aboptinsnpbrowser

Related article can be found on: Page 38

INFO

52BiosystemsSolutions

customer focus

Genomics

SpotlightDr Raija L.P. Lindberg, Head of the Clinical

Neuroimmunology Laboratory, The Department of

Research and Neurology, University Hospital of Basel

“Our aim is also to be able to estimate the response of various treatments and,

in that way, to optimise patient management and hopefully prevent

long-term damage in the central nervous system”

he main topics of work at the Clinical Neuroimmunologylaboratory are analysis and research on multiple sclerosis(MS). The aim of our work is to comprehensively define

altered physiological pathways at various stages of development of multiple sclerosis (e.g. during the development of lesions, during relapses and remissions) in various tissue compartments. We approach this by studying the gene expression patterns in brain tissue and peripheral blood from MS patients, and in brain and lymph node tissues in two animal models of MS (delayed-type hypersensitivity (DTH) and experimentalautoimmune encephalomyelitis (EAE).

Our research is currently following two lines of enquiry. The first aim is to get better insight into the pathogenic molecularmechanisms of MS by comparing the gene expression profiles of MS tissues to that of normal, non-neurological patients. Defining altered pathways in the disease will ultimately lead to newtreatment strategies of MS. Secondly, we are looking for predictiveand diagnostic markers for the disease itself and its course. Our aim is also to be able to estimate the response of varioustreatments and, in that way, to optimise patient management andhopefully prevent long-term damage in the central nervous system.Ideally, these markers should be expressed in easily accessiblespecimens, e.g. blood or urine, rather than for instance incerebrospinal fluid (CSF). Part of our research programme is linkedto clinical trials for candidate drugs and treatment regimens.

What populations are you using for your studies?

This laboratory is closely connected to the MS clinic in BaselUniversity Hospital. My colleagues are mostly clinicians who aretreating MS patients daily and consequently we have good access topatient samples to use in our research. We are also studying autopsysamples from MS patients and comparing them with samples from other neurological diseases and control subjects.

We aim to verify the findings from the human studies with thecorresponding animal models of MS and test possible new treatmentstrategies first with animal models before starting clinical trials.

T

What are your preliminary findings?

MS is characterised by the formation of disseminated areas ofdemyelination and neuronal damage, called lesions or plaques,therefore it has been defined as a focal disease. We have studied theexpression profiles not only in lesions but also in so-called normalappearing white matter (NAWM), which is located in a vicinity of lesions but looks macroscopically normal. Our main findingsfrom these studies provide molecular evidence of a continuum ofdysfunctional homeostasis and inflammatory changes in lesions andNAWM, and support the concept of MS as generalised as opposedto a focally restricted disease of the CNS. Moreover, our findingscorroborate that even in the progressive phase of disease, reparativesystems are active to compensate for structural damage.

What do these studies involve?

We are using a microarray technique to get comprehensiveinformation of the gene expression profiles altered in MS. In parallel, we are using real-time PCR analysis to validate themicroarray findings and further evaluate the expression ofindividual target genes.

And this is where TaqMan® Gene Expression Assays

from Applied Biosystems come in?

Yes. I saw an advertisement shortly after the products becameavailable and realised that it was exactly what I needed at this stageof the project. Technically, the dynamic measuring range ofmicroarrays is limited and may fail to detect subtle changes forgenes with very high or very low expression levels. Therefore weused quantitative real-time PCR (RT-PCR) and the TaqMan GeneExpression Assays to further verify leads from the array data.

What did you do before the TaqMan Gene Expression

Assays became available?

I either had to design the assays myself or run a pre-developed assayreagents (PDAR) system if it was available. The problem is thatdesigning assays from scratch is so time-consuming and with thetime saved by the TaqMan Gene Expression Assays, I can get onwith the data analysis itself. In the early stages, not all the targets we needed were available but most of the important ones were and while I worked on these, the others were being developed. More assays have become available and now all I need to do is tobrowse the list for product numbers of genes of interest and thenjust order. Unfortunately, at the moment all the assays are for thewell-defined sequences. For ESTs (expressed sequence tags) andother unknown sequences I still need to design assays myself.

What direction will your research now take?

At the moment we are using human assays as much as possible andthese are readily available as TaqMan Gene Expression Assays.However, we will soon need to confirm that mouse and/or ratmodels show the same phenomenon as we have seen in humans.New possible treatment strategies need to be first tested in animalmodels before proceeding to clinical trials. For that we will needrodent alternatives to the TaqMan Gene Expression Assays. Assays for other species are now becoming available, therefore wewill probably combine TaqMan Gene Expression Assays andcustom TaqMan Gene Expression Assays.

“More assays have become available and now all I need to do

is to browse the list for product numbers of genes

of interest and then just order”

For more information on:ABI PRISM® 7000 Sequence Detection System enter 128

For more information on:TaqMan® Universal PCR Master Mix enter 129

For more information on:TaqMan® Gene Expression Assays enter 130

INFO

Above: Dr. Raija L.P. Lindberg

“The aim of our work is to comprehensively

define altered physiological pathways at

various stages of development of multiple

sclerosis in various tissue compartments.”

Dr. Lindberg gained a PhD in Biochemistry at theUniversity of Turku, Finland, in 1986. From 1987 to 1990, she was Visiting Research Associate in theLaboratory of Reproductive and DevelopmentalToxicology, NIEHS, NIH, North Carolina, USA,where she specialised in molecular biology. From 1990to 1998, as an independent Research Associate inDepartment of Pharmacology, the University of Basel,Switzerland, she developed and studied a mouse modelfor the human disease, Acute Intermittent Porphyria.From 1998 to date she has been Head at the ClinicalNeuroimmunology Laboratory at Department ofResearch and Neurology in Basel.

Technologies used:

» ABI PRISM® 7000 Sequence Detection System

» TaqMan® Universal PCR Master Mix

» TaqMan® Gene Expression Assays

BiosystemsSolutions53

n January 2004, Applied Biosystems formally introduced

the 8500 Affinity Chip Analyzer for antibody

characterisation and protein interaction applications.

This instrument uses SpotMatrix SPR technology to enable

label-free measurement in real-time for kinetic analysis of

biomolecular interactions.

The first 8500 system was sold to Genentech (San Francisco,

CA) in January, which they have used to investigate various

protein-protein and protein-peptide interactions. Their research

using the 8500 Affinity Chip Analyzer was presented at two

recent conferences:

IBC’s Advances in Protein Science:

APS 2004 World Summit

» April 19-21, 2004 at the Boston Park Plaza, Boston, MA

CHI’s Beyond Genome 2004:

Proteomics - Addressing Challenges in Proteomic Analysis

» June 23-24, 2004 at the Fairmont Hotel, San Francisco, CA

Applied Biosystems will also be presenting data generated from

the 8500 Affinity Chip Analyzer at conferences worldwide.

Please contact your Applied Biosystems representative for

more information on this technology.

I

54BiosystemsSolutions

Research using the8500 Affinity Chip AnalyzerPresented at two recent conferences

bio-highlights

BiosystemsSolutions55

bio-highlights

etween 15 December 2003 and February of this year we carried out a market research survey to discover more about our customers' Real-time PCR

needs. First of all, we would like to take this opportunity to thank all our customers who returned their completedquestionnaires. The valuable information that we received was very much appreciated and will help us better match yourfuture Real-time PCR needs!

All the participants of the survey, who completed and returnedtheir questionnaire, had the chance to enter our prize draw to winone of five Casio Exilim Z4 digital cameras.

The draw took place in February, and the five lucky winners werepresented with their digital cameras in March. We would like tocongratulate the following lucky customers and hope they havefun with their new cameras:

Dr Susana Benlloch, Hospital General, Alicante, Spain

Mr Jérome Vicenzi, Cypher Science/TraceTag Europe, Paris, France

Dr Alessandra Movilia, Ospedale Civile di Legnano, Milan, Italy

Mr. Ludo A.B. Oostendorp, Streeklaboratorium voor de Volksgezondheid voor Groningen en Drenthe, Groningen, The Netherlands

Dr. Olga Slamborová, KlinLab s.r.o, Prague, Czech Republic

B

Applied Biosystemsasks...what are your Real-time PCR needs?

AB-ONLINE >>>News on demand for integrated science

elcome to e-News, our on-line news channel.Providing an exciting mix of features and links, e-News is a valuable source of information for any

researcher working in the life science arena.

Each issue of e-News provides you with the latest innovations inyour area of interest, together with applications focused news tohelp move your research forward.

» Application Focus: Provides the latest news on application techniques and market focus

» Product Focus: Updates on our latest product releases» Special Offers: Details of our current special offers » Events: Updates on our forthcoming events in Europe» Customer Relations: Features life science news plus links

to more information, support and training

To register for Small Molecules e-News visit:http://news.appliedbiosystems.eu.com/LCMS_eNews

To register for Proteomics and Cell Biology e-News visit:http://news.appliedbiosystems.eu.com/Pro_CB_eNews

To register for Assays e-News visit:http://news.appliedbiosystems.eu.com/assay_enews

To register for Forensics e-News visit:http://news.appliedbiosystems.eu.com/forensic_enews

To register for Genomics e-News visit:http://news.appliedbiosystems.eu.com/genomics_enews

W

bio-highlights

56BiosystemsSolutions

Applied Biosystems now offers two new tools to

significantly ease the work and to shorten the

time between hypothesis and result

he breadth of genomic information now availablemeans a huge amount of the researcher’s time isspent in gathering and organising this information.

In mutation detection particularly, literature and databasesearches are necessary before concentrating on the intendedresearch project.

After collecting background information on the genes or the pathway of interest, setting up the experimental design requires diving into the literature and performingvalidation experiments.

Applied Biosystems now offers two new tools, VariantSEQr™

Resequencing System1 and myScienceSM, to significantly easethe work and to shorten the time between hypothesis and result.

VariantSEQr Resequencing System

A complete application solution for detecting mutations orvariants in thousands of disease-related human genes.

» Accomplishing all resequencing projects with superior data quality

» No primer design and validation processes required

» Identifying all variants using the most accurate technology available

» Ready-to-use primer sets formulated at the working concentration and analysis software that is fully integrated with Applied Biosystems reagents and instruments

» Reducing the complexity of data analysis with relevant information such as the reference sequence for all amplicons provided with the analysis software

Confidence values are assigned by a primer design algorithmand take problematic areas into account (low complexityrepeat, GC content, GC count in localised regions, propensityto form triple helices etc.). Each RSA (ResequencingAmplicon) is designed to perform according to the assignedconfidence value when using recommended protocols andApplied Biosystems equipment and reagents.

myScience

A free-of-charge online life science research environment forgenomic-based experiments is saving your time by providingall necessary information in one place2. With a single query, it allows you to gather critical information about a gene orpathway with a direct link to a broad offering of informationconnected to this gene:

» Finding relevant information on NCBI* and other online information e.g. LocusLink genes, RefSeqs and NCBI mRNAs for Human or equivalents for Mouse and Rat, plus additional content such as Celera’s protein classification information for these genes and articles from key researchers*National Center for Biotechnology Information

» Easy-to-use graphical map viewer allows to visualise information from a single gene up to a whole chromosome

» Applied Biosystems’ genomic products, such as VariantSEQr Resequencing System can easily be identified and selected based on the provided information

What information related to VariantSEQr is

available in myScience, and how do you find it?

Searching

There are four different ways to search for specificVariantSEQr Resequencing Systems:

Search can be done by Keyword, Batch ID code numbers (e.g. RefSeq and GenBank accession numbers), ChromosomeLocation or by Molecular Function

Result list (RSS ID)

The Search Results (figure 1) are displayed as basicinformation about the Resequencing Primer Set (RSS), like gene identification information, type, size, number ofamplicons and percentage of coverage. More detailedinformation about the gene is just one click away.

Detail Report Gene

From the Detail Report Gene page, a hyperlink “GeneName/Gene Symbol /ID” provides general information aboutthe gene, which would normally be collected by in depthliterature search, for example: » Involvement in processes and gene function

» Background information about location and category of the specified gene

» Hyperlinks to NCBI reports

T

Narrowing in on your mutations

INFO

For more informationvisit:http://myScience.appliedbiosystems.com

For more information on:VariantSEQrResequencing Systementer 131

AcknowledgementsBeate Rätz, Heinz Doersam,Applied Biosystems, Germany

Figure 1.

The assay results page contains detailed information about the Resequencing Set

BiosystemsSolutions57

A graphical view of the chromosomal region can be found usingthe ‘map view’ hyperlink on that page.

Detail Report Resequencing Primer Set (figure 2)Information about the resequencing primer itself set can beaccessed via hyperlink ‘RSS ID’.

Information about the amplicons in the set includes coordinates,covered region, and a confidence value.

The information on the RefSeq public gene assembly and theCelera Discovery System gene assembly of a specific gene ismapped to a supertranscript called Target Region, including thebase sequence of that region. This information can be viewed in agraphical display (figure 3).

With the ‘Export Results’ link the myScience research results datacan be saved into a text file to be exported to the researcher’scomputer, for archiving or further studies.

How to order VariantSEQr Resequencing Sets?

If you are registered and logged in at our online store(www.appliedbiosystems.com/catalog) you can directly order theselected VariantSEQr Resequencing Sets from myScience.

» Simply check the box in front of a Resequencing Primer Set and click add to selected Shopping Basket

» Prices will reflect all your special conditions

» You will receive an immediate order confirmation

» You can track your shipments and order history online

Check out for the current VariantSEQr promotion at myScienceand get a 20% discount together with other attractive offers.

References

1. Mutation Analysis made easy, Biosystems Solutions Issue 8, 2003

2. Walk along the Chromosomes, Biosystems Solutions Issue 9, 2004

Discover Your Gene Research Tools

on myScience today.

You are just a click away.

http://myScience.appliedbiosystems.com

Figure 2.

Detail Report resequencing primer set

Figure 3.

Target Regions and RSA (resequencing amplicons) are depicted in separate tiers with the Gene or RefSeq transcript as a reference

pplied Biosystems Mass Spectrometry systems are helping

scientists solve analytical problems with Mass Spectrometry

in clinical trial, food/beverage, and environmental

applications and bring unparalleled experience and product quality to

the broad field of testing, identification and quantification. To support

this we have produced a comprehensive Application Binder focusing

on Food and Environmental testing:

» The analysis of polycyclic aromatic hydrocarbons (PAHs) by LC/MS/MS using an atmospheric pressure photoionisation source

» Detection of acrylamide in starch-enriched food with HPLC/MS/MS

» Simultaneous analysis of multiple anti-bacterial drugs in food products using LC/MS/MS

» Simultaneous determination of residues of approximately 100 pesticides and metabolites in fruit and vegetables by LC/MS/MS

» Determination of organophosphorous and organonitrogen pesticides in fruit and vegetables by LC/MS/MS

» HPLC/MS/MS analysis of bitter acids in hops and beer

» Isolation and characterisation of anti-viral nucleoside compounds from the Chinese herb taraxacum mongolicum

» Determination of N-nitrosamines in baby bottle rubber teats by liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry

» Fast multi-residue pesticide analysis in soil and vegetable samples

» The combination of monolithic columns and modern HPLC/MS/MS instrumentation for high chromatographic throughput and sensitive detection of beta-agonists

» Determination of polar organophosphorus pesticides in aqueous samples by direct injection using HPLC/MS/MS

» Detection of nitrofurane metabolites in food with HPLC/MS/MS

» Simultaneously characterising and quantifying chloramphenicol and its metabolite using LC/MS/MS

» High sensitivity quantitation of metabolites of nitrofuran antibiotics in animal tissue using LC/MS/MS

To download any of the application notes listed above visithttp://www.appliedbiosystems.eu.com/mk/get/ABSMPDFREG

A

LCMS Food and EnvironmentalApplications Binder

58BiosystemsSolutions

bio-highlights

bio-highlights

BiosystemsSolutions59

Applied BiosystemsCustomer Training Courses

July

Aug

Sep

tO

ct

Call Jonathan MacBeath 01925 282530

email: [email protected]

Course July August Sept Oct

ABI PRISM® 7000 Sequence Detection System 05-06 21-22 Reply no. 132

ABI PRISM® 7700 Sequence Detection System 01-02 07-08 Reply no. 133

ABI PRISM® 7900 Sequence Detection System 12-13 11-12 Reply no. 134

Applied Biosystems 7300 & 7500 Real-Time PCR Systems 29-30 19-20 30-01 Oct Reply no. 135

ABI PRISM® 310 Genetic Analyzer 26-27 28-29 Reply no. 136

ABI PRISM® 3100 Genetic Analyzer 02-03 08-09 05-06 Reply no. 137

16-17

Applied Biosystems 3730 DNA Analyzer 01-03 Reply no. 138

SeqScape™/GeneMapper® Software 04 & 18 10 07 Reply no. 139

HID Course 15-17 13-15 Reply no. 140

Call Birgit Berenz +49 (0)6151 9670 5251

email: [email protected]

Course July August Sept Oct

API 2000™/API 3000™ Systems* 08-09 05-06 23-24 14-15 Reply no. 141

API 4000™ System 06-07 03-04 21-22 12-13 Reply no. 142

Q TRAP® System - Small Molecules 26-28 16-18 13-15 18-20 Reply no. 143

QSTAR® System - Small Molecules 07-09 15-17 Reply no. 144

4000 Q TRAP™ System 28-30 18-20 15-17 20-22 Reply no. 145

Q TRAP® System - Proteomics 10-12 12-14 Reply no. 146

QSTAR® XL System - Proteomics 03-05 26-28 Reply no. 147

Voyager-DE™ PRO/STR Workstation 27-29 14-16 Reply no. 148

BioCAD® Workstation/Vision™ Workstation Dates on Application Reply no. 149

* = 2 days training in-house + WebEx follow-up afterwards

Applied BiosystemsLingley House120 Birchwood BoulevardWarrington, CheshireWA3 7QH TEL: 01925 825650 FAX: 01925 282502

http://europe.appliedbiosystems.com

European Sales Offices

Austria Tel: +43 (0)1 867 35 75Belgium Tel: +32 (0)2 532 44 84Denmark Tel: +45 45 58 60 00Finland Tel: +358 (0)9 693 794 27France Tel: +33 (0)1 69 59 85 85Germany Tel: +49 (0)6151 96 700Italy Tel: +39 039 83891Luxembourg Tel: +31 (0)180 392 400The Netherlands Tel: +31 (0)180 392 400Norway Tel: +47 23 16 25 75Portugal Tel: +351 22 605 33 14Spain Tel: +34 91 806 1210Sweden Tel: +46 (0)8 619 4400Switzerland Tel: +41 (0)41 799 77 77United Kingdom Tel: +44 (0)1925 825650

European Managed Territories

Africa Tel: +27 11 478 0411Czechia Tel: +420 2 3536 5189 Hungary Tel: +36 1 471 89 89Poland Tel: +48 22 866 4010 Russia Tel: +7 095 775 1166S.E. Europe, Middle East,West Asia Tel: +44 (0)1925 282481

iScience. To better understand thecomplex interaction of biological systems,life scientists are developing revolutionaryapproaches to discovery that unitetechnology, informatics, and traditionallaboratory research. In partnership withour customers, Applied Biosystemsprovides the innovative products, services,and knowledge resources that make thisnew, Integrated Science possible.

Biosystems Solutions

E d i t o r i a lApplied Biosystems, Lingley House, 120 Birchwood Boulevard, Warrington, Cheshire WA3 7QH, UK.Tel: +44 (0)1925 825650 Fax: +44 (0)1925 282502 email: [email protected]

E d i t o rKay L Hill

C o n t r i b u t o r s – A p p l i e d B i o s y s t e m sPeter Boogaard, Heinz Doersam, Andy Felton, Chris Grimley, Verena Guhl, Jean-Luc Gy, Tony Hardware, Martin Heinrich, Martin Hornshaw, Karsten Lueno, Wolfgang Mayser, Sabine Motor, Pierre Paroutaud, Victoria Parr, Rosi Santner, Vince Woodall

D e s i g n MacRae Communications Ltd, 1 Belgreen House, Green Street, Macclesfield, Cheshire SK10 1JH, UK.Tel: +44 (0)1625 869689 Fax: +44 (0)1625 511678 email: [email protected] Web: www.macraemarketing.com

Applera Corporation is committed to providing the world's leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses.

For Research Use Only. Not for use in diagnostic procedures. The PCR process and the 5' nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd.

The ABI PRISM® 3100 and 3100-Avant Genetic Analyzers and the Applied Biosystems 3730 and 3730xl DNA Analyzersinclude patented technology licensed from Hitachi, Ltd. as part of a strategic partnership between Applied Biosystems and Hitachi, Ltd., as well as patented technology of Applied Biosystems.

T r a d e m a r k sABI PRISM, Analyst, Applied Biosystems, BigDye, BioBeat, FMAT, GeneMapper, GeneScan, iTRAQ, NanoMate, PROCISE,QSTAR, SeqScape, SNaPshot and SQL*LIMS are registered trademarks and AB (Design), Applera, API 2000, API 3000, API 4000, BioTrekker, Celera, Celera Discovery System, Genotyper, iScience and iScience (Design), myScience, NanoSpray,PANTHER, SNPbrowser, SNPlex, TOF/TOF, Tempus, TURBO V and Voyager are trademarks of Applera Corporation or itssubsidiaries in the U.S. and/or certain other countries.

AmpliTaq Gold, GeneAmp and TaqMan are registered trademarks of Roche Molecular System, Inc. ICAT is a registered trademark of the University of Washington, exclusively licensed to Applied Biosystems Group of Applera Corporation.

Q TRAP is a trademark of Applied Biosystems/MDS SCIEX instruments, a joint venture between Applera Corporation and MDS Inc.

Applied Biosystems/MDS SCIEX is a joint venture between Applera Corporation and MDS Inc., the instrumentation technology division of MDS Inc.

All other trademarks are the property of their respective owners.

Information is subject to change without notice.No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any other means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the copyright holder. Copyright rests with the publisher.

©2004 Applied Biosystems. All rights reserved. Printed in The Netherlands 05/04.

Visit our Online Store at:

https://store.appliedbiosystems.com