proteomics - 123seminarsonly.com€¦ · proteomics september 2004 6th melbourne bioinformatics...

Andrew Clippingdale, LICRProteomics

September 20046th Melbourne Bioinformatics Course

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Proteomics:Background and Applications

Andrew B. Clippingdale

Joint Proteomics Services Facility, Ludwig Institute for Cancer Research and the Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia

[email protected]

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Definition of Proteomics



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Talk Summary

• Quantitative Proteomics Using 2D-Gels

• Proteomics using Multidimensional LC

• Protein Complex Analysis by Proteomics

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“I would like to analyse differential protein expression (or degradation) in disease.

Would using proteomics help?”



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Normal Colon

Adenoma (polyp)

Carcinoma

Development of Colon Cancer

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pH3 pH10

+ - --

- -+

-+

+

+-+ -

++

+++ Time0

+---

- - -+

+

++ -+

+++ ++ +

+ - --

-

++ -+ Time1

-+ Time2

1st dimension separation

Isoelectric Focusing of Proteins



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Second

dimension

separation


Molecular Weight Separation of Proteins

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Second

dimension

separation


Sample 2D-Gel



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2DE of Differential Protein Expression

Conventional 2-D

Are spot differences real?

differences

control gel 1

stain

treated gel 2

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“Ok, that gives me a start. What about gel to gel variability? Can we quantitate those changes?”



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2D DIGE

Dual scan

Dye 1

Dye 2

control

treated

Ettan™ DIGE System - 1

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Control sample Treated sample

Perfect Spot Overlays



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Ettan™ DIGE System - 2

Protein extract 1label with Cy3

Pooled internalstandard label with Cy™2

Protein extract 2label with Cy5

Mix labelled extracts

2DEseparation

Cy5

Typhoon™Variable Mode

Imager

Cy3

Cy2

DeCyder™ Differential AnalysisSoftware

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“How do I identify the proteins?”



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MS Protein Identification Types

• Peptide mass fingerprinting (PMF)

• Primary sequence analysis (MSMS)

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UV Laser λ 337 nmAnalyte + Matrix

MALDI-TOF-MS - 1

+ + +

++++

++ ++ +

+



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LaserPrecursor ions

Chargedfragments

LinearMode

Laser ReflectronMode

MALDI-TOF-MS - 2

Resolution

Sensitivity

Throughput

Sequencing Capability

Excellent

Excellent

Excellent

Poor

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Peptide Mass Fingerprinting

Sequence Coverage: 31%

Score: 129 (Score > 74, p<0.05)



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“But What if I Can’t Use 2D-Gels?”

• Low abundance proteins not visualised

• Resolution of target proteins unsatisfactory

• Membrane proteins

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Talk Summary

• Quantitative Proteomics Using 2D-Gels





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• Identification of molecules released locally or into the circulation by CRC cells could be exploited as:– - Diagnostic (non-invasive kit)– - Prognostic– - Therapeutic

In vivo

-Identify proteins differential expressed from the blood of normal and CRC subjects

Albumin54.31%

Immunoglobulin G16.61%

α1-Antitrypsin3.83%

α 2-Macroglobulin3.64%

Immunoglobulin A3.45%Transferrin3.32%

Haploglobin2.94%Immunoglobulin M1.98%

Other9.91%10%

α1-Acid glycoprotein1.25%

Complement C31.12%

Hemopexin1.05%

α2HS-Glycoprotein0.80%

α1-Antichymotrypsin0.58%

α-trypsin inhibitor0.58%

Gc-Globulin0.48%

Ceruloplasmin0.48%

Complement C40.45%

Fibronectin0.42%

Prealbumin (thyroxine-binding)0.32%

C1 Esterase inhibitor0.32%

α1B-Glycoprotein0.29%

β2-Glycoprotein I0.29%β2-Glycoprotein II

0.27%Complement C1

0.22%

Remaining1%

Clinically useful protein amounts vary by ten orders of magnitude

Human Plasma

Composition of Human Plasma

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Margin-bufferreservoirs Peristaltic

pump

Counter-flowreservoir

Separationchamber

7Electrode-buffer

reservoir

Electrodebufferpump

Sampleinlet ports

Main port

Counter-flow solution96-Channeltubing outlet

96-Well plate

96-Well plate

Electrodecompartment

Electrodecompartment

Running-bufferreservoir +pH 3 pH 10 -

+

+ -

-

Agilent 1100 HPLCOctopus™ Free-Flow Electrophoresis

96-well plateautosampler

96-well platefraction collector(s)

Columncompartment

Binary pumping system

UV diode-arraydetector

Fluorescencedetector

Solvent degassing system

2D-Liquid Chromatography



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4 12 20 28 36 44 52 60 68 76 84 92

0

10

20

30

40

50

60 0

Elu

tion

(% O

rgan

ic s

olve

nt)

Well number

0.2Intensity O.D. (215nm)

2

4

6

8

10

12

pH (

)

2D-LC of Human Plasma

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Fig[MS].9

Electrospray Ionization (ESI)



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Quadrupole Ion Trap (Tandem MS)

Ion Accumulation Precursor Ion Isolation Collision Induced Dissociation Mass Analysis

A. B. C. D. E.

Ion Accumulation Precursor Ion Isolation Collision Induced Dissociation Mass Analysis

A. B. C. D. E.

Resolution

Sensitivity

Throughput


Poor

Good

Medium

Good

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m/z

Rel

ativ

e In

tens

ity %

y 7

b2 y 6

b3 y 5

b4 y 4

b5 y 3

b6 y 2

y 1b7

b ions contain theN-terminus of the peptide

y ions contain theC-terminus of the peptide

Fragmentation of Protonated Peptides



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Protein identification(ranked output)

m/z

Simulate MS/MS Spectra

MS/MS Spectrum

Pattern matchingIle-Asn-Val-Gln-Lys

Ala-Arg-Glu-Leu-Ile

Search database forPeptides of same mass

Cys-Arg-His-Trp

Ala-Ile-Thr-Ala-Leu-Leu

Ile-Asn-Val-Gln-Lys

Database Searching using MSMS Data

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4 12 20 28 36 44 52 60 68 76 84 92

0

10

20

30

40

50

60 0

Elu

tion

(% O

rgan

ic s

olve

nt)

Well number

0.02Intensity O.D. (215nm)

2

4

6

8

10

12

pH (

)1

3

1619

1815 11

12

1

10

92

8

4

145 6

7

1720

13

Proteins Identified from Human Plasma - 1



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1. α1-acid glycoprotein2. α1-anti-trypsin3. α1anti-chymotrypsin4. α1B glycoprotein5. Apolipoprotein AI6. Apolipoprotein AII7. Antithrombin III8. complement C4A/B9. ceruloplasmin10.Fetuin A

11. αFibrinogen12. Fibrinogen13. Fibrinogen gamma chain14. Haptoglobin15. Haptoglobin-216. Human serum albumin17. Immunoglobulin18. Kininogen19. Serotransferrin20. Transferrin

Proteins Identified from Human Plasma - 2

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0 1 2 3 4 5 6 7 8

0.00

0.05

0.10

0.15

0.20

Abso

rban

ce a

t 215

nm (

)

Retention Time (min)

RPHPLC Profiles of Fraction 43

Unadulterated PlasmaMulti-affinity bound

Multi-affinity unbound



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“This will test you! I’m studying membrane protein complexes with some subunits which may

not be in the database.”

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Talk Summary

• Quantitative Proteomics Using 2D Gels





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NADH:Ubiquinone oxidoreductase46 subunits, Mr > 106

Fearnley, I.M. et al, (2001) JBC 276, 38345-38348Carroll, J.G. et al, (2002) JBC 277, 50311-50317

Mitochondrial Complex 1

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1DE of Complex 1 and Subcomplex 1λ



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51 kDa49 kDa

42 kDa

75 kDa

30 kDa

24 kDaTYKYASHI

SGDH

B13GVRT

B18

PGIV

B9

KFYI

51 kDa

39 kDa

PDSW + B22

PGIV

B18

GVRTB13

SGDHPSST

B14

B8

MNLL

B9

KFYI

MWFEMLRQ

AQDQ

B17.2

SAES

B14.5b & B12

PFFD B17B16.6

B14.5aB15unknown

pH 3 pH 10 pH 6 pH 11

TricineSDS/PAGE

2D-Gel of Bovine Complex 1

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REFLECTRON

ELECTROSPRAYSOURCE

QUADRUPOLE 1

QUADRUPOLE 2 GAS COLLISION CELL

DETECTORPUSHERTOF

Q-Tof™ High Resolution MS-MS

Resolution

Sensitivity

Throughput


Excellent

Excellent

Medium

Excellent



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100 200 300 400 500 600 700 800 900 1000 1100 1200 1300M/ z0

100

%

(185.98) Y K R L V T K (113.02)y Max

965.32y6864.30

y5603.72(M+2H) 2+

487.70a7 2+

281.11205.06y188.03

343.14b2

765.26y4

604.22652.21

y3766.26 865.30

947.32

966.32

967.33 1093.39y7 1152.17

(Asp-N digest)

Acetylated N-terminal Peptide of B14.7

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Homologue of N. crassa subunit 21.3b

B14.7 Nucleotide and Amino Acid Sequence



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Homologue of Human GRIM-19

B16.6 Sequence and Alignment

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Proteomics:

•Various protein chemical techniques

•Targeted and global studies

•Unique insights into complex systems



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Joint ProteomicS LaboratoryLudwig Institute for Cancer Research and the Walter and Eliza Hall

Institute of Medical Research, Parkville, Victoria 3050

Richard SimpsonRobert Moritz

David Frecklington

MRC-Dunn Human Nutrition UnitWellcome Trust/Medical Research Council Building, Hills Road,

Cambridge CB2 2XY, UK

Ian FearnleyJoe Carroll

Acknowledgements

proteomics - 123seminarsonly.com€¦ · proteomics september 2004 6th melbourne bioinformatics...

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