Dr. Khalid Rehman HakeemFellow ResearcherFaculty of Forestry

Universiti Putra Malaysia (UPM)Serdang-43400, Selangor

MALAYSIA

Plant Proteomics

Presently Fellow Researcher (Associate Prof. Research) @ Universiti Putra Malaysia

Books with international Publishers

Our Latest Proteomics Research

For full publication list visit my Research gate account https://www.researchgate.net/profile/Khalid_Hakeem

ORPersonal website

www.Khalidhakeem.weebly.com

Omics (IF=2.77) ABAB (IF=1.89) Mol. Breeding (IF=3.25)

ABAB (IF=1.89) IJMR (IF=2.47)

Contents•Introduction •Techniques involved•Protein isolation•Sample preparation•First dimension: Isoelectric focusing•Second dimension: SDS-PAGE•Detection of protein spots: staining •Imaging analysis & 2D Gel databases•Spot handling: excision, in gel digestion•Mass spectrometry•Software analysis•Applications•Challenges

Science 291 (2001) 1221.

WHAT IS PROTEOMICS?

The analysis of complete complements of proteins: identification and quantification; modifications; interactions; and activities. FUNCTION.

AND HOW DO THESE CHANGE DURING A BIOLOGICAL RESPONSE?

Peck SC (2005) Update on Proteomics in Arabidopsis. Where do we go from here? Plant Physiol 138: 591-599

Major Proteomics Directions

Adapted from Human Proteome Organization (www.HUPO.org)

PTMs

Rose et al.(2004) Plant J 39: 715-733.

“Proteomics is an increasingly ambiguous term being applied to almost any aspect of protein expression, structure or function.”

Tools of Functional Genomics

Colebatch et al (2002) Functional Genomics: tools of the trade. New Phytol 153: 27-36.

•Whole Genome Sequence –complete, but does not show how proteins function or biological processes occur

•Post-translational modification –proteins sometimes chemically modified or regulated after synthesis

•Proteins fold into specific 3-D structures which determine function

•Gain insight into alternative splicing

•Aids in genome annotation

Why Proteomics ?

Solving the Puzzle of Protein Function

Proteomics is a multipotent tool central to research efforts in many fields and disciplines. Maximum functional utility will come from joint efforts.

A Single Gene Can Produce Many Proteins

Peck (2005) Plant Physiol 138: 591

Targeting sequence

Principle:

One gene ≠ one transcript ≠ one protein

ONE Genome but MANY Proteomes!

Proteomics and genomics are inter-dependent

Genome Sequence

mRNA

Primary Protein products

Functional protein products

Determination of gene

Genomics

Proteomics

Proteomics

Protein Fractionation

2-D Electrophoresis

ProteinIdentification

Post-TranslationalModification

MALDI-TOF @ Indian Institute of Integrative Medicine ,Jammu.

( Applied Biosystems ,Model 4800 )

Mascot search engine (http://www.matrixscience.com)

Basic Proteomic Analysis Scheme

Protein Mixture

Individual Proteins

Blast & Mascot analysis

Protein identification

Protein extraction and sample preparation

Normal Stressed

Reduction and alkylation

SDS-PAGE (2nd Dimension), Visualization (Coomassie BB/Silver staining)

HPLC/Mass spectrometry and protein identification

Isoelectric focusing (1st Dimension)3…pH…10+ - + -3…pH…10

3…pH…10(kDa) M

100

30

10

3…pH…10(kDa) M

100

30

10

LS-MS peptide-mass fingerprint spectrum of the tryptic digest of differential protein and its idenification on MASCOT

absent

Over-expressed

induced

under-expressed

Spot numbering (1, 2, 3….x), excision and digestion

Plant Material

Pro

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, res

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The goal of two-dimensional electrophoresis is to separate and display all gene products present.

* It is the only method currently available which is capable of simultaneously separating thousands of proteins.

* The first dimension of 2-DE - isoelectric focusing (IEF). - pH gradient support -pI

* TheSecond dimension of 2-DE - SDS as surfactant. - Molecular mass.

•High resolution from independent protein parameters.

*In the early 1970s, first use of 2-DE to separate serum proteins.

The second dimension of 2-DE - sodium dodecyl sulfate PAGE (SDS-PAGE).

•A critical step in 2-DE.

Solubilization, denaturation, reduction & removal of non-protein sample components.

Optional modifications for more proteins displayed, shorter focusing time, and more sharply focused spots:

- Chaotropes: 8M Urea 2M Thiourea & 7M Urea- Surfactants: 4% CHAPS 2% CHAPS & 2% Sulfobetaine 3-10- Reducing agents: 100mM DTT 2mM TBP (Tributyl phosphine) &/or 65mM DTT

Sample preparation

Separating the Proteome• The protein genome is separated by several

different methods.

• Many researchers are first separating portions of the genome, such as isolating organelles, and then analyzing that portion.

• This is because often proteins of interest,

regulatory proteins are in low abundance.

• The most commonly used method is 2-dimensional gel electrophoresis. – Consists of using isoelectric focusing with

SDS polyacrylamide gel electrophoresis

Isoelectric focusing • This separates proteins based on isoelectric point

• The isoelectric point is the pH at which the protein has no net charge.

• pH gradients may be large 2-10 or small 6-7 (Optimum=4-7)

• Typically this is done with an immobilized pH gradient gel strip or with a tube gel containing a low concentration of polyacrylamide.

• Ampholytes are added to create a pH gradient in an electric

field and the proteins are loaded.

• The IEF gel is placed in an electrophoresis system for up to 24 hours and the proteins form tight bands at their isoelectric point.

• The IEF gels are now ready for the second method.

Figure is from “Principles of Biochemistry” Lehninger, Fourth Edition

SDS Polyacrylamide Gel Electrophoresis • The second dimension separates the proteins based on size.

• There are two parts, the stacking gel which concentrates the sample and the running gel that is used to separate the proteins.

• The IEF gel is soaked in a solution containing chemical to denature the proteins including sodium dodecyl sulfate (a detergent which gives the proteins a net negative charge). This means that all proteins will move in one direction.

• The IEF gel is then put in the one long well in the stacking gel, sealed in place with agarose, and the proteins subjected to an electric field to separate.

• The larger proteins are found at the top and the smaller ones are found at the bottom of the gel.

2-Dimensional Gel Electrophoresis• In a 2D gel the proteins appear as spots on the

gel rather than bands. These spots can then be further processed or used for mass spectrometry directly.

• Further processing usually includes spot excision, trypsin digestion, and mass spectromety

• Analysis may also include differential 2D gel electrophoresis

– In this case a control and sample are separately labeled with a fluorescent molecule.

– The samples are mixed and electrophoresed in the same gels.

– A laser scanner is used to identify each spot and a program puts the two images together.

2-D Gel Electrophoresis

Visualization of proteins

Coomassie blue staining

Detect 36-47ng

Silver staining

Detect 0.5-1.2ng

Fluorescent staining

Detect 1-2 ng

From Jefferies, et al., http://www.aber.ac.uk/parasitology/Proteome/Tut_2D.html#Section%201

Images fromhttp://www.kendricklabs.com/2d+CoomassieBlue.htm

http://www.unil.ch/dbcm/page48211_fr.html

Alternate Separation Methods• The first dimension is run in larger

agarose tube gels with ampholytes. – This has less resolution than

polyacrylamide gels. The tubes are sliced and the proteins are allowed to diffuse out.

• Gel regions are cut, proteins eluted and the proteins are then separated by capillary electrophoresis.

• Capillary electrophoresis has a much greater resolution for the proteins mass.

• Proteins are eluted from the capillary in the process and can be collected. They are readily available for mass spectrometry.

Alternative Separation Methods• Whole proteome is analyzed at once.

• Proteome is digested with protease (trypsin)

• Digested proteome is injected to HPLC with 2 columns in series (mixed bed ion exchange and reverse phase)

• Peptides are eluted from ion exchange onto reverse phase and then separated on reverse phase column.

• Peptides then enter ESI-MS-MS

PD Quest Software

•Spot picking (gel excision) – manual or mechanical ?

Fuji Science Amersham

Mass Spectrometery

• Separates ions based on mass to charge ratio.– Charges are placed on the protein or the peptide by

ionization.

• Two most common types of ionization are:• Matrix-Assisted Laser Desorption Ionization.– MALDI causes fragmentation of the protein during

ionization. Can be used to get more information about the fragments. Easier to do than ESI.

• Electrospray ionization (ESI)– ESI can give whole protein masses as well as complex

masses. If the proteins is first separated by reverse phase HPLC before injection only the subunits masses will be known.

Matrix-Assisted Laser Desorption Ionization (MALDI)

• MALDI causes fragmentation of the protein during ionization.

• It Can be used to get more information about the fragments. Easier to do than ESI.

• Requires sample to be placed in matrix that absorbs appropriate wavelength light.

• Matrix generates heat and forms ions of matrix and what is around it.

“MALDI-TOF” mass spec

• Matrix-Assisted: Your favorite molecule is co-crystallized (dried) with a light-adsorbing compound (“matrix”).

“MALDI-TOF” mass spec

• Laser Desorption/Ionization: A pulse of laser light is used to force molecules into gas phase and ionize them.

+

+ ++ ++ +

MALDI-TOF mass spectrometry

• Ions are then accelerated in a high-voltage electrostatic field…

++ + +++ +

(+)

(-)

MALDI-TOF mass spectrometry

…and allowed to drift down a long high-vacuum flight tube.

• At the end of the tube, ions strike a detector.

• Drift time (time of flight) is measured by sophisticated electronics.

++ + ++

+ +

(-)

HOW IT WORKS ?

Electrospray Ionization

Mass Analyzers

• Important parameters– Sensitivity• How few ions can be detected.

– Resolution• How well different masses can be determined.

– mass accuracy• How reproducible and correct are the masses.

Interpreting a mass spectrum• x-axis is…

– tof–m / z– “mass” of ion

• y-axis is…

–“hits” on the detector

–number of ions detected 365.0 760.6 1156.2 1551.8 1947.4 2343.0

Mass (m/z)

0

4.5E+4

0

10

20

30

40

50

60

70

80

90

100

% Intens

ity

Spec #1 MC[BP = 1053.6, 44590]1053.6285

656.1051

869.4992

1117.6282

1550.87361072.6033 1355.7562549.3439 822.4886

1426.8657 1690.9995617.4088

1566.8917

916.5234 1036.59751890.0891

591.3327 741.4249 1569.9111893.4636585.3485 1075.62562008.27261443.80271278.6373679.4092537.3699 1088.6108919.5099 1674.6634800.4719 1436.7851 1893.11101252.6669 2033.17691003.8684 1625.0525 2185.28701432.1174 1856.6127527.2609

TOF~velocity~acceleration~mass

• The relationship of TOF vs MASS is calibrated via co-analysis of standards whose masses are known.

2D-PAGE Analysis Software

• 2D-PAGE technology has been in use for over 20 years, and potentially provides a vast amount of information about a protein sample.

• However, due to difficulties with data analysis, it remains only partially exploited.

Analysis problems

• It can be very difficult to compare the results of two experiments to yield a differential expression profile:

• Can be severe warping of gel due to– uneven coolant flow– voltage leaks– tears in gel

• Can be problems with normalisation of– background– spot intensity

• Can be differences in sample preparations.

Current state of software

• Correct identification and alignment of spots from the two gels has generally been a process with a lot of manual intervention - hence very slow.

• The processing power available with today’s PCs means that automated analysis is starting to become possible.

• One vendor claims a throughput of 4 gel pairs per hour can be compared and annotated by an experienced user of their package.

Automated gel matching

• Gel matching, or “registration”, is the process of aligning two images to compensate for warp.

• Some packages still require the user to identify corresponding spots to help with gel matching.

• The Z3 program from Compugen has a fully-automated gel matching algorithm:– define set of small, unique rectangles.– compute optimal local transformations for rectangles.– Interpolate to make smooth global transformation.

• Note that this makes use of spot shape, streaks, smears and background structure, which other programs discard.

Spot detection

• Once the gel images have been matched, the program automatically detects spots. Algorithms are generally based on Gaussian statistics.

Spot Quantitation

• The positions of detected spots are calibrated to give a pI / mW pair for each protein.

• A value for the expression level of the protein can be calculated from the overall spot intensity.

• Some programs do not quantitate each gel separately, but calculate relative intensity pixel by pixel. This may be a more accurate approach.

Differential Expression

• The user can set threshold values for the detection of differential expression. This helps reduce the amount of information displayed at once.

• In this example, a protein expressed only in the second sample is circled in red. The yellow circles show proteins which are differentially expressed.

Annotation

• Some systems allow semi-automatic annotation of spots, based on a database of proteins listing their pI / mW values.

• Proteins of interest can also be excised from the gel and sent on to mass spectrometry for definitive identification. The ProteomeWorks system from Bio-rad offers such an integrated solution for 2D-PAGE and MALDI.

Multi-experiment Analysis

• One useful feature of modern programs is the ability to collate data from many runs of the same experiment.

• Spots which only appear in one gel are likely to be artifacts, and are removed from the analysis.

• This is an excellent way to reduce noise and enhance weak signals.

Links

• Z3 system (Compugen) - http://www.2dgels.com/• Melanie3 (SIB) - http://us.expasy.org/melanie/• ProteomWeaver (Definiens) -

http://www.proteomweaver.com/• PDQuest (Bio-Rad) - http://www.biorad.com/• Delta2d (Decodon) - http://www.decodon.com/

Applications of Proteomics

• Protein Profile- A Global view of proteins of Interest

• Differential display- Biomarker discovery

• Protein post translational modification- Protein Function

Biological Applications• Proteome maps

– starting point for major study in genomics– questions of interest:– How much of the genome is transcribed and translated in the living

organism?– What effect different growth conditions have on the proteome?

• studies are going on in– Eukaryotes like Humans

• extensively modify their proteins by N- or C-terminal cleavages• decorate them with sugars and/or phosphates, sulfates... PTMs• Yeast Saccharomyces cerevisiae, ....• Fruit fly Drosophila melanogaster, ....• Plant Arabidopsis thaliana, ....

Biological Applications, cont.

• Tracking complexity– host-pathogen or host-parasite interactions

• nitrogen fixation in legumes by association with bacteria (Rhizobium) to form nodules

• infection of flax by flax rust• Immunogetic proteins

– identifying proteins from infectious disease agents recognised by the immune system

– vaccine candidate for microbial pathogens (e.g. Chlamydia trachomatis infection)

– allergy research: which grass pollens are most immunogenic?– identification of allergens (proteins) in Latex (gloves) by a 2D PAGE

run using Latex as a sample!

• Improved agricultural products

– engineering resistance to pathogens/parasites into various plants

– most of these resistance mechanisms involve expression of toxic or

protective proteins

– discovery of new toxic or protective proteins

– wool proteome project to investigate economically important characters

like colour and fibre strength

• Value added agricultural products

– remanufacture low value products

• proteinaceous whey as a by-product of cheese manufacture

• investigation whether this whey can be used to grow recombinant

bacteria for biotechnological production

Biological Applications, cont.

• Quality control

– Is the hamburger mince sold as beef really beef or a mixture of beef and kangaroo or even buffalo?

– proteome technology brings precision and definition to a new level in protein-based products

• For further applications please refer to the ProXPRESS PIP on the intranet

– forensic sciences

– microbiology

– epidemiology

– taxonomy

Biological Applications, cont.

Proteomics contribute to Target Identification and Validation

HUMANGENOME

DISEASEASSOCIATIONS

PUTATIVETARGETS

TARGETFUNCTIONIN CELL

BIOLOGICALLYVALIDATEDTARGETS

HITIDENTIFICATION

HITS TOLEADS

LEADOPTIMISATIONTOXICOLOGY

EFFICACY/ P.O.C. DEVELOPMENT

Knowledge of direct interactionspartners, location, expression &modifications helps describe aprotein’s function within the cell

Network of interactions andpathway expression predictstoxicology, required drugcharacteristics and side effects.

Toxicology and Pharmaceuticals

• Multiple overlapping pathways are influenced by toxins or drug treatment

– simultaneous identification, characterization and quantification of numerous of gene products and their PTMs

– massively parallel approach offered by Proteomics

• Retinoic acid

– used in dermatology and onco-haematology

– retinoic acid acylation of proteins (PTM)

– detection of this protein retinoylation with proteomics

• Phosphorylation

– “on” or “off” signals of biochemical pathways by kinases and phosphatases, complex networks

• etc....

Cancer• Carcinogenic products act similarly to

pharmaceutical agents, affecting the PTMs and

the level of expression of numerous proteins

– oncogene product alterations & cell cycle specific

protein modifications play important role in

tumorgenesis and cancer progression

• studies are going on in

– brain, thyroid, breast, lung, colon, kidney, bladder,

ovary, bone marrow

Rice genotypes (G) 1 2 3 4 5 6 7 8 9 10 ----------------------------------20

Assays for N-use efficiency

G6cv. Rai Sudha

High NUE

G8cv. Munga Phool

Low NUE

Proteomics analysis for differential NUE

N N N NN-use efficient proteins

qRT- PCR conformation

Working out the Nitrogen Efficient mechanism in rice and wheat genotypes using proteomics

Cadmium toxicity induced alterations in the root proteome of green gramin contrasting response towards iron supplement

Results suggest that green gram plants exposed to cadmium stress are able to change the nutrient metabolic balance in roots, but in the mean time regulates cadmium toxicity by iron supplements.

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Sowbiya Muneer†*, Khalid Rehman Hakeem†*, Rozi Mohamed and Jeong Hyun Lee.Int. J. Mol. Sci.2014,15, 1-x manuscripts doi:10.3390/ijms150x000x

On going Proteomics projects @ UPM

•Mapping the proteome of thick-walled and rapidly growing bamboo for the development of thick walled bamboo plantlets. 2014-15 (118,000 RM)…..Continue

•Proteomic identification of gaharu systnesis enzymes in pathogen-induced Aquilaria for the production of high-impact compounds. (340,000 RM)…….Submitted

•Plant diversity, Ecosystem Dynamics and Proteomics of Matang Mangrove Forest, Taiping, Perak. (300,000 RM)…….Submitted

Challenges for 2D Technology

• Whole cell extracts are too complex. How can complexity be reduced?

• Abundant & soluble proteins are easily characterized.How can rare and membrane proteins be found?

• Large format gels are difficult to handle and slow. Is there an alternative?

Challenges for 2D Technology

• Abundant & soluble proteins are easily characterized.How can rare and membrane proteins be found?

•

• Increase sensitivity by fluorescent dye labelling! Use ProXPRESS for gel imaging!

Challenges remain with Gels

• Gels & staining limit S/N, dynamic range

– today’s standard staining methods

• silver staining: 10 to 70 -200 ng/spot (enough for MS)

• Coomassie Blue: less sensitive

• fluorescent dyes expectations: 0.1 ng to 10 mg/spot (necessary

for faint protein amounts)

• Gels can be selective

• Membrane proteins need special conditions

• Low copy number proteins are missed

• fluorescent dyes expectations: 0.1 ng to 10 mg/spot (necessary

for faint protein amounts)

82

QUESTIONS AND COMMENTS ARE

WELCOME