1 Dunn Color

8/3/2019 1 Dunn Color

1/16

1

EDUCATION SESSION

Overview of Proteomic Technologies

Mike DunnProfessor of Biomedical Proteomics

Proteome Research CentreUCD Conway Institute

Dublin, Ireland

Expression proteomicsDifferential display or discovery proteomics

Global analysis of changes in protein expression during abiological process or in disease

Cell mapping proteomicsInteractomics

Study of protein interactions

1. Protein-protein interactionsIdentify members of functional protein complexes

Pathway (e.g. signaling) analysisAnalysis of protein networks

2. Protein-small molecule interactionsProtein-drug binding

Proteomics

Analysis of complex mixtures of proteins

Differential analysis of multiple samples

Quantitation of proteins present in samples

Identification and characterisation ofproteins present in samples

Bioinformatics(identification, annotation, dissemination)

Two-dimensional gel electrophoresis(2-DE) of intact proteins

Tried and trusted technology

MS-based (gel-free) methodsShotgun LC-ESI-MS/MS of

total tryptic digest of proteinsQuantitation by stable isotope labelling

(e.g. SILAC, ICAT, iTRAQ)

Protein chipsIntact proteins

(e.g. SELDI-MS, protein, tissueand antibody arrays)

Choice of proteomics platformpI

MWt

High-resolution 2-DE is > 30 years old!

First dimension isoelectric focusing (IEF)Separation of proteins according to their charge (pI) under

denaturing conditions

Second dimension SDS-PAGESeparation of proteins according to their size (Mr) underdenaturing conditions

Protein separation and display: Two-dimensional

polyacrylamide gel electrophoresis (2-DE)

Still the method of choice for the majorityof differential protein expression

studies of disease

2-DE is currently the only technique thatcan be routinely applied for parallel quantitative

expression profiling of large sets of complexprotein mixtures such as whole cell

and tissue lysates(Goerg, Weiss and Dunn, Proteomics 2004, 4: 3665)


2/16

2

pI

MrX 103

-still the method of choice for most proteomic projects

- well established method (IPG-IEF vs. SDS-PAGE)

- can separate 2,000 to 5,000 proteins on a single gel

-IPG-DALT gives highly reproducible 2-DE proteinprofiles

-proteins can be visualisedat high sensitivity

-computer analysis of differential protein expression

-interfaces with MS protein identification methods

Protein separation and display: Two-dimensionalpolyacrylamide gel electrophoresis (2-DE)

IEF (charge)

SDS-PAGE (size)

1. Sample preparation

2. Separation of proteins by 2-DE

3. Sensitive detection of the protein profile

(silver staining, fluorescent staining, DIGE)

4. Quantitative computer image analysis to detect

differentially expressed proteins

5. Protein identification

(Western immunoblotting, Edman degradration,amino acid compositional analysis, MALD-TOF MS,

ESI-MS/MS)

2-DE proteomics workflow

Sample preparation

No universal methodNeeds to be optimised for each sample type

General recommendations

Keep it simple to increase reproducibility

Minimise possibility of protein modifications

that can lead to artefactual spots on 2-DEDo not heat samples containing urea

Protein carbamylation by isocyanate ions results inmultiple spots (charge trains) on 2-DE

Proteolysis: Keep samples cold and

add protease inhibitors

Sample preparation

Body fluids(Serum, plasma, urine, CSF, BAL, saliva, tears etc)

May need to concentrate and remove salt (e.g. urine)Analyse directly after dilution with solubilisation buffer

Cell and tissue samplesUsually disrupted and solubilised in lysis buffer

SolubilisationDisrupt all non-covalently bound protein complexes and

aggregates into a solution of individual polypeptides

Chaotrope + Detergent + Reducing agent

(9.5 M urea, 4% NP-40, 1% DTT)

IEF with immobilised pH gradients (IPG)Wide range, medium range, narrow range

The first dimension

Potential to match IPG profile with thedistribution of sample protein pI values

Second dimension SDS-PAGELaemmli discontinuous Tris/glycine

buffer system usually used(15-250 kDa proteins)

Tris-tricine buffer system for betterseparation of smaller (3-20 kDa) proteins

Borate buffer system forhighly glycosylated proteins


3/16

3

Body fluid: Human plasma(SWISS-2DPAGE: http://www.expasy.org/swiss-2dpage/) Body fluid: human urine

Mr

pI

Uromodulin

Michelle Downes, UCD Conway

Cells: Human microvascular endothelial cells

Ciara McManus: UCD Conway Institute

IPG 4-7 L

SDS-PAG

E

Tissue: Human heartIPG 3-10 NL

12%

SDS-P

AGE

Streaking ofbasic proteins

3 acidic/neutral 7 basic 10

2-DE of basic proteins

Reducing agentsRequired to disrupt protein inter- and

intra-molecular disulphide bonds

But DTT that is normally usedis protonated at high pH and migrates to

the anode during IEF

Results in re-oxidation of S-S bonds

Poor separations of basic proteins (pI > 7.5)due to streaking of spots

2-DE of basic proteinsSolution

Samples must be cup loaded at the anode

Replace DTT with HED (hydroxyethyldisulphide)DeStreak reagent (GE Amersham)

Oxidises cysteinyl groups to form mixed disulphideswhich prevent reformation of disulphide bonds

Less streaking and improved resolutionof basic proteins by 2-DE

(Prot - S

+ R- S - S - R Prot - S - S - R + R - S)


4/16

4

2-DE of basic proteinsHuman heart proteins

Mr

pH 3-10 pH 6-11

Mr

In-gel rehydration Cup-loading at anode

with DeStreak

Good separation of

basic proteins

Streak due toproteins with pI 10,000 proteins

Dynamic range of protein abundance106for cells and tissue, 1010for body fluids

Need to study sub-proteomesZoom (narrow range) 2-D gels

Cellular, sub-cellular and protein fractionation

2-DE of membrane proteins

Alternative reagents for solubilisation

Chaotropes: 7 M urea, 2 M thiourea

Detergents: - Zwitterionic detergents (e.g. CHAPS)

- Linear sulphobetaines (SB3-10, ASB 14)

- Triton X-114 phase partitioning (Wissing et al, 2000)

- SDS pre-solubilisation and detergent exchange

Reducing agents: Tributyl phosphine, TCEP, DeStreak (Amersham)

Consider using alternative 2-D gel system for membrane proteins16-BAC/SDS-PAGE (Hartinger et al, 1996)CTAB/SDS-PAGE (Navarre et al, 2002)(Doubled) dSDS-PAGE (Rais et al, 2004)1-D SDS-PAGE (combine with LC-MS/MS analysis of gel bands;

GeLC-MS/MS)

16-BAC/SDS-PAGE

First dimension

Molecular weight separation in an acidic discontinuous PAGE systemStacking gel: pH 4.1; separating gel pH 2.1Cationic detergent: benzyldimethyl-n-hexadecylammonium chloride (BAC)

Second dimension

Molecular weight separation by standard SDS-PAGE

Proteins have slightly different migration properties in the two systems,allowing a two-dimensional separation


5/16

5

Mitochondrial membrane proteins(S. cerevisiae)

57 proteins identified by LC-MS/MS

16-BAC/SDS-PAGE Problems associated with 2-DE

Protein solubility

Membrane proteins under-represented on2-D gels due to solubility problems

Proteomic coverage2,000 proteins separated by 2-DE

Sample may contain >10,000 proteins




2-DE can only separate 2,000 proteins per gel

Increased separation distance40 x 40 cm gels using CA-IEF (Klose 1999)

(5,000 proteins)

IPG-IEF strips up to 24 cm are available24 x 21 cm format

(2 to 3,000 proteins)

Use of overlapping narrow range IPGsZoom gels

Virtual increase in separation area

3-10NL

3.5-4.5L 4.5-5.5L 5.5-6.7L

5-6L4-5L

4-7L 6-9L

Zoom gels(narrow range IPGs)

Increased proteomiccoverage

6-9L

pI 4.0 5.0 6.0 9.0Mr

42.0

27.2

18.7

14.7

288 1325 568

Total number of spots = 2178

Mr

pI 3 10

42.0

27.2

18.7

14.7

Number of

spots = 1141

Zoom gels(narrow range IPGs)

Increased proteomiccoverage

Mr

pI 3 10 4 7 5.5 6.7

42.0

27.2

18.7

14.7

27.227.2

27.2

A B C

1 1 2 1 2 3

EH or Hsp27 1 EH & 2 Hsp27 1& 2 EH, 3 Hsp27

5.81 5.78 5.81 5.81 5.83 5.85pI

Westbrook et al, Electrophoresis 2001 22: 2865

Separation of Enoyl-CoA-hydratase and Hsp 27


6/16

6

Problems associated with 2-DE

Protein solubility

Membrane proteins under-represented on2-D gels due to solubility problems






Protein detection/visualisationDynamic range 102 to 104

Organic dyes Silver Radioactivity

CoomassieBrilliant Blue(CBB) R-250

(100 ng)

Colloidal CBBG-250 (10 ng)

alkaline silver diamineor acidic silver nitrate

(1 ng)

Fluorescent stains

- SYPRO Ruby- SYPRO Red

- SYPRO Orange- Cy2, 3, 5 (pre-electrophoretic)

(1 ng)

(Very high

sensitivity)

Sensitivity

Dynamic range

Linearity

Compatibility with MS

Protein detection/visualisation

Current limit of detection around 1 ng

Can detect proteins present in total cell extractat around 10,000 copies/cell

Most regulatory proteins < 10,000 copies/cell

But can investigate downstream effectsof their action

Sub-proteomics may allow proteomic analysisof low copy number proteins

Problems associated with 2-DE

Protein solubilityMembrane proteins under-represented on

2-D gels due to solubility problems






Sample fractionation and enrichment

Cell and sub-cellular fractionation

Immuno-isolation

Electromigration analysis (e.g. FFE)

Flow cytometry

Classical density gradients (organelles)

Sequential/selective extraction

Laser capture microdissection

Selective solubilisation of membrane proteins(Triton X-114 phase separation)

At low concentration Triton X-114 binds tothe cell membrane by partitioning into thelipid bilayer

Membrane bilayer is disrupted and lysed asdetergent concentration increases abovecritical micelle concentration (CMC)

At CMC lipid-protein-detergent mixedmicelles are produced

A further increase in detergent concentrationwill result in a heterogeneous complex ofdetergent, lipid-detergent and protein-detergent micelle formations

Triton X-114 mimic's the lipid bilayerenvironment incorporating the disassociatedprotein into micelle formation

Micelles formed are analogous to the bilayerof the biological membrane

Partitioning of membrane bound and solubleproteins into detergent and aqueous phases


7/16

7

250 mg of tissue ground in liquid nitrogen

10% Triton X-114 + cold PBS

Overnight at 4C

37C for 30min

30min spin @ 5000g

30min spin @ 20,000g

AQ DT

10% Triton X-114 Ice cold PBS

37C for 15min- spin 37C for 15min- spin

x3

Acetone precipitation- Rapigest

ME

TH

OD

ME

THO

D

Membrane and mitochondrial proteindistribution (human heart) (LC-MS/MS)

AQ Phase

20%

80%

Membrane Non-Membrane

DT Phase

62%

38%

Membrane Non-membrane

AQ Phase

14%

86%

Mitochondrial proteins Non-mitochondrial proteins

DT Phase

50%

50%

Mitochondrial proteins Non-mitochondrial proteins

McManus et al, unpublished

Laser capture microdissection Laser microdissection of human heart toobtain cardiac myocytes and blood vessels

2-DE protein profile

Mrx 103

220

97

66

45

30

20.1

14.3

IPG 3-10 NL IPG 3-10 NL

Cardiac Myocytes Blood Vessels

1

2

3

5

4

1 = cardiac tropomyosin; 2 = cardiac light chain I; 3 = cardiac light chain II;4 = smooth muscle transgelin; 5 = smooth muscle tropomyosin

Protein fractionation and enrichment

Electrophoresis in solution(e.g. IEF, FFE, MCE)

Chromatography(IEX, SEC, AC)

Immunoprecipitation

Isolation of interacting protein complexesInteractomics


8/16

8

Anderson and Anderson, Molec. Cell. Proteomics 2002 1: 845

Interestingproteins

10 proteins 12 proteins

Human plasma

Increasing proteomic coverage

Affinity depletion of albumin, Ig andother major plasma proteins

Fractionation of plasma proteins(LC, IEF, etc.)

Analysis (2-DE or MS/MS platforms) ofindividual fractions

1D SDS PAGE Analysis of Depleted SerumAgilent Multiple Affinity Removal System (MARS6)

Jennifer Byrne: UCD Conway Institute

205

11697

84

66

5545

36

29

24

20

14. 2

IgG Light chain

HSA/ Antitrypsin/ Haptoglobin

Haptoglobin

IgG Heavy chain/ Antitrypsin

HSA/ IgATransferrin

Crude

SerumBound Depleted

serum

-1-antitrypsin

Albumin

IgGIgA

-1-antitrypsinTransferrinHaptoglobin

(85% of total protein)

pH 4 7

Albumin

Transferrin

IgGheavy

chain

IgGlight

chain

Haptoglobin

Antitrypsin

IgA

50g of crude human serum run on pH 4-7 IPG strips.


pH 4 7

50g of depleted human serum run on pH 4-7 IPG strips. Serum was depleted using the

Agilent Multiple Affinity Removal System (MARS6), a polyclonal antibody-based depletion

system that removes the 6 most highly abundant proteins.


2-DE Analysis of Depleted Serum

Undepleted

Depleted

Overlay

DeCyder analysis:

850 spots in undepleted serum1527 spots in depleted serum76% increase in spot number660 additional low abundance spots


9/16

9

Serum depletion

Now a variety of columns and spin cartridges available, e.g.

Agilent:Human MARS 6,7, 14; Mouse 3

Sigma-Aldrich:Human Proteoprep 20

Beckman Coulter:Human IgY-12,; Rodent IgY-7

670 L sample/chamberTotal: 1-2 mg protein

Zuo & Speicher (2002) Proteomics 2: 58

Human serum: Increasing proteomic coverageFractionation by solution phase IEF

(Invitrogen ZOOM IEF Fractionator)

Fractions of

human plasmaon 2-DE

zoomnarrow pH range

IPG gels

Unfractionated

pH 3-4.6 / IPG 3-4.5 pH 4.6-5.4 / IPG 4-5 pH 4.6-5.4 / IPG 5-6

IPG based IEF OFFGEL fractionation

with 0.1 0.6 pH resolution

Fractionation of proteins or peptides

In-solution fraction recovery foreasy transfer to 2-DE or LC-MS

Two power supplies allow simultaneousfractionation of two sample sets withbroad differences in concentration

Up to 16 samples fractionated atthe same time

g to mg load capacity

Agilent 3100 OFFGEL Fractionator

Michel, P. E., Reymond F. Arnaud I. L., Josserand J., Girault H. H. and Rossier J. S. (2003)Protein fractionation in a multicompartment device using Off-Gel isoelectric focusing.Electrophoresis 24, 3-11.

(a)

(b)

(c)

(d)

Agilent 3100 OFFGEL Fractionator Agilent 3100 OFFGEL Fractionator


10/16

10

Computer analysis

Quantitative computer image analysis to detectdifferentially expressed proteins

Commercial 2-DE analysis softwarerunning on desktop workstations

(PC, Mac, Unix)

Progenesis (Non-linear Dynamics)DeCyder (Amersham)

PDQuest (Bio-Rad)Melanie / ImageMaster (GeneBio, GE Healthcare)

Kepler (LSB)

Z3 (Compugen)ProteomWeaver (Definiens)

Major bottleneck in proteomic analysis

Computer analysisMajor bottleneck in proteomic analysis

filtering,

spot detection quantitative analysis ofdetected proteins

gel matching

Solubilise

Cy3 label

Solubilise

Cy5 label

Combine

for 2-DE

Image

fluorescence

Differential

quantitation

Cy3 Cy5

2-D Fluorescence Differential In-Gel Electrophoresis(ETTAN 2-D DIGE: GE Healthcare)

Control Disease

Typhoon

fluorimager

Overlay of images

But still need to compare multiple pairs of samples run on

multiple 2-D gels: problems of inter-gel normalisation

Solubilise

Cy3 label

Solubilise

Cy5 label

Combine

for 2-DE

Image

fluorescence

Differential

quantitation

Cy3 Cy5

Control Disease

Standard pool

Solubilise

Cy2 label

Cy2

2-D Fluorescence Differential In-Gel Electrophoresis(ETTAN 2-D DIGE: GE Healthcare)

Use of Cy2-labelled pooled standard to

normalise Cy3 and Cy5-labelled spotsbetween gels

Not normalised to standard

GE Healthcare

Normalised to standard


11/16

11

Altered left ventricular proteins in dilated cardiomyopathy

Significant alterations in abundance of 93 proteins in DCM

More proteins decreased (80) than increased (13) in abundance

Protein identification

Mass spectrometry

- Western immunoblotting- Protein sequencing (Edman degradation)

- Amino acid compositional analysis

Peptide mass fingerprinting

by MALDI-MS

Partial amino acidsequencing by

ESI-MS/MS

orMALDI-MS/MS

high sensitivity & throughput

automation (spot cutting,

digestion, target spotting)

quantitation

de novosequencing

characterisation of post-

translational modifications

(phosphorylation,

glycosylation,etc)

bioinformatics

data handling

Peptide mass fingerprinting by MALDI-MS

Nucleotide/protein

sequencedatabase

Excise spotand

digest withtrypsin

Digestdatabasein silico

with trypsin

Massspectrum

byMALDI-MS

Theoreticalmass

spectrum

Identityor

Homology

Peptide mass fingerprinting by MALDI-MS

Peptide mass fingerprinting by MALDI-MS Peptide mass fingerprinting by MALDI-MS


12/16

12

Peptide mass fingerprinting by MALDI-MS Peptide sequencing by ESI-MS/MS (CID)

Peptide sequencing by ESI-MS/MS (CID) Peptide sequencing by ESI-MS/MS (CID)

Two-dimensional electrophoresis (2-DE)of intact proteins


MS-based (gel-free) methodsShotgun LC-ESI-MS/MS oftotal tryptic digest of proteins

Quantitation by stable isotope labelling(e.g. ICAT, iTRAQ, SILAC)




MWt

Gel-free Proteomics

Multi-dimensional protein identification technology(MudPIT)(Link et al, Nature Biotechnology 1999, 17: 676)

Strong cation exchange (SCX) LC

coupled either on- or off-line withRPLC-MS/MS


13/16

13

Alternatives to SCX separation

SDS-PAGE: GeLC-MS/MS

SDS-PAGE of sample (usually mini-gel format)Slice unstained gel into 20-30 slices

Digest in situ with trypsinEach digest analysis by RP-LC-MS/MS

Provides MWt information on sample proteins

Slice and dice

J Prot Res 2006, 5: 2071

Alternatives to SCX separation

OffGel Fractionator

24 cm pH 3-10 IPG with 24 wells

pI can be used as a parameter inpeptide/protein identification

Immunodepleted human serum

24 Offgel fractions

versus50 SCX fractions

OGE identified:2x more proteins3x more peptides

Agilent: Poster at 2006 BSPR/EBI Meeting (Hinxton, UK)

Analysis of single mouse brain2 x 30 SCX Fractions

60 RPLC-MS/MS runs

Relative abundance of proteins in the mouse brain:Good correlation between the protein

concentration (g/uL) and thenumber of LC-MS/MS spectra (spectrum count)

used to identify each protein

Very challenging to analyse large numbers of samples

A quantitative method is required for differential expression studies:e.g. stable isotope coding (e.g. ICAT, iTRAQ)

Analysis of adult mouse ventricle4 x Sub-cellular fractions

MudPIT: On-line SCX-RP-LC-MS/MS5 repeat runs of each fraction

J Am Soc Mass Spectrom 2005, 16, 12071220

Challenging to analyse large numbers of samplesQuantitative method is required for differential

expression studies:Stable isotope coding (e.g. ICAT, iTRAQ)

Julka S and Regnier FE (2005)Briefings in Functional Genomics and Proteomics 4: 158

In vivolabelling methodsSILAC

In vitrolabelling methodsICAT (proteins labelled -> Cys peptides analysed)

16O/18O (labelled during proteolysis)iTRAQ (tryptic peptides labelled)

SILAC(Stable Isotope Labeling with Amino acids in Cell culture)

Mol Cell Proteomics. 2002 May;1(5):376-86

(Ong et al, Mol Cell Proteomics 2002, 1: 376)

e.g. L-leucine and deuterated L-leucine

SILAC is only applicable to cultured cells

and cannot be used fortissue and body fluid samples


14/16

14

Isotope-coded affinity tag (ICAT) reagents(Gygi et al, Nature Biotechnology 1999, 17: 994)

Isotope-coded affinity tag (ICAT strategy)(Gygi et al, Nature Biotechnology 1999, 17: 994)

Molecular & Cellular Proteomics 2:299314, 2003

Original deuterated ICAT reagent too large and interfered with MS fragmentation

Cleavable ICAT reagent developed

Iodoacetamide and biotin groups separated by spacer containing nine13C atomsBiotin tag removed from coded Cys peptides by acid hyrolysis

iTRAQ Reagents

Globally code N-termini and Lys residues of all peptides in

a tryptic digest through acylation with N-methyl hydroxysuccinamide esters of N-methyl piperazines

4 iTRAQ reagents, so can multiplexup to 4 different samples

(8-plex iTRAQ reagents now available)

Label-free methods for quantitative MS

Stable isotope labelling is expensive

Label-free methods:

1. Ion intensity: Mass spectral peak intensities ofpeptide ions correlate well with proteinabundances in complex samples

2. Spectral counting: Compares the number of

MS/MS spectra assigned to each protein and thiscorrelates well with protein abundances incomplex samples

Two-dimensional electrophoresis (2-DE)of intact proteins


MS-based (gel-free) methodsShotgun LC-ESI-MS/MS ofTotal tryptic digest of proteins

Quantitation by stable isotope labelling(e.g. ICAT, iTRAQ, SILAC)




MWt


15/16

15

SELDI-MS (Ciphergen)(Merchant & Weinberger, Electrophoresis 2000, 21: 1164)

Good for liquid samples such as body fluidsFavours proteins and peptides


16/16

Protein arrays

High density protein arrays using cDNA expressionlibraries

Applications:1. Protein-antibody interaction analysis

Characterisation of binding specificity of antibodiesScreening serum or plasma for autoantibodies

2. Protein-protein interaction analysis

Network analysis3. Protein-chemical compound interaction analysis

Drug development4. Enzyme-substrate activity analysis

Phosphatases, peroxidases, galactosidases,restriction enzymes, protein kinases

Leuking, Cahill and Muellner, DDT 2005, 10: 789

Protein arrays

Protein arrays

Profiling of the autoantibody repertoireof plasma from patients with dilated

cardiomyopathy (DCM) against a humanprotein array consisting of 37,200

redundant, recombinant humanproteins

1 Dunn Color

Documents

Transcript of 1 Dunn Color