Proteomics and mass spectrometry

Manimalha Balasubramani

Outline

Mass spectrometers Protein identification Quantitative proteomics Protein-protein interactions

A mass spectrum

799.0 1179.2 1559.4 1939.6 2319.8 2700.0

Mass(m/z)

6.3E+4

0

10

20

30

40

50

60

70

80

90

100

% I

nte

nsi

ty

1258

.560

3

1303

.700

7

878.

4913

983.

4860

1031

.537

4

1254

.561

4

1114

.542

8

1657

.795

3

842.

4926

1586

.806

4

1232

.590

7

1964

.888

2

924.

5113

1153

.533

4

965.

4456

1035

.569

6

833.

0566

1315

.578

012

80.5

370

1074

.540

5

2120

.988

3

1360

.720

9

1191

.613

0

1689

.786

5

870.

5201

2518

.106

2

1800

.932

4

1395

.706

2

1848

.941

9

1475

.737

4

1630

.773

8

2211

.052

0

1593

.769

3

2393

.082

3

2169

.920

7

2439

.087

2

2021

.911

6

Basically measures mass

Adapted from google

Components…

Adapted from an Analytical chemistry textbook

Ionization process

ESIESI

Nobel prize in Chemistry, 2002

Matrix Assisted Laser Desorption Ionization

ElectroSpray Ionization

MALDIMALDI

MALDI – Matrix Assisted Laser Desorption Ionization

ESI – Electro Spray Ionization

Mass analyzers – several designs

Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207

GPCL inventory ABI Voyager DE PRO, walk-up use ABI 4700 Proteomics Analyzer Thermoelectron LCQ Deca with Surveyor

HPLC ABI Qstar Elite with Ultimate 3000 HPLC Bruker micrOTOF with Ultimate 3000 HPLC Bruker 12 Tesla FTMS with Ultimate 3000

HPLC

Time-of-flight (TOF) analyzers

MALDI TOFVoyager DE PRO

ESI TOFUltimate 3000 with micrOTOF

MALDI TOF - principle

KE = zeV = 1mv2

2

MS of serum albumin

MALDI TOF

ESI TOF

Tandem mass spectrometer

MALDI TOF/TOF

MS and MS/MS

Ion Trap

MS, MS2, MS3, ….MSn

Quadrupole-q-TOF

ESI QqTOF

…installation phase….

FT MS

…bottom line…

..Resolution and mass accuracy…..Resolution and mass accuracy…

FWHMFull width at half maxima of a peak

Resolution and mass accuracy

R = M Δm

R = resolutionM = mass of the peak of interest Δ m = width in daltons of the peak

Δm measured at 50% peak

height is the Full Width at Half

Maxima (FWHM)

Mass accuracy is measured as parts per million value

ppm = 106Δm = 106 M R

outline

Mass spectrometers Protein identification Quantitative proteomics Protein-protein interactions

Peptide Mass Fingerprinting - PMF

Database entry NCBI

From: http://gobi.ym.edu.tw/course/mass/2004-0325.pdf

Informatics Search engines

Mascot, Matrix Science Sequest, Thermoelectron

Free-ware Protein prospector (http://prospector.ucsf.edu/) TPP tools

(http://tools.proteomecenter.org/TPP.php)

Database searching using MASCOT

Overview of the experimentSubmission of data to MASCOT

webserver

1D SDS PAGE of proteins

Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207

Mass spectrum

699.0 1159.2 1619.4 2079.6 2539.8 3000.0

Mass (m/z)

1.6E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 Reflector Spec #1 MC=>TR[BP = 1479.9, 15779]

1479

.882

4

1439

.896

7

1567

.827

6

1163

.700

0

2045

.127

3

927.

5582

1881

.022

3

1724

.927

2

1305

.788

8

1730

.772

3

1399

.775

1

1249

.695

4

1895

.038

6

1283

.788

1

1433

.807

4

1554

.743

7

1640

.027

7

841.

5205

2555

.290

3

1763

.782

0

1687

.869

1

2262

.055

7

1516

.713

5

1014

.682

7

1590

.861

9

1081

.547

9

1121

.552

0

2458

.305

2

1195

.624

3

789.

5378

898.

5428

2493

.350

1

Mass to charge ratio (m/z)

Inte

nsity

Peak list Compiled from the mass spectra

Mass list Mass list and intensity

Submitted to the search engine

http://www.matrixscience.com/

Mascot scoringA frequency factor matrix, F, is created, in which each row represents an interval of 100 Da in peptide mass, and each column an interval of 10 kDa in intact protein mass. As each sequence entry is processed, the appropriate matrix elements fi,j are incremented so as to accumulate statistics on the size distribution of peptide masses as a function of protein mass. The elements of F are then normalised by dividing the elements of each 10 kDa column by the largest value in that column to give the Mowse factor matrix M:

After searching the experimental mass values against a calculated peptide mass database, the score for each entry is calculated according to:

Where MProt is the molecular weight of the entry and the product term is calculated from the Mowse factor elements for each match between the experimental data and peptide masses calculated from the entry.

List of common contaminants Trypsin autolysis peptides Matrix peaks Keratin from skin, hair Other contaminants

Protein Identification

Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207

Tandem mass spectrum

http://qbab.aber.ac.uk

Tandem mass spectrum

69.0 386.8 704.6 1022.4 1340.2 1658.0

Mass (m/z)

3105.9

0

10

20

30

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% Int

ensit

y

4700 MS/MS Precursor 1570.7 Spec #1 MC[BP = 175.1, 3106]

175.1326

1056.51071554.7853

1571.9679684.3845

1556.5172

112.0977 1558.4042813.4371

246.1672 333.21051559.94171441.7213

480.27491039.4810316.1747 1570.2634741.3559463.2531 627.3450 942.4836120.0979 1040.9976 1551.70021268.54271171.5131400.2173229.1560 758.3326490.3423 629.3128 910.867972.1029 1445.2834837.0470

Database Searching•Peptide Mass Fingerprinting•Sequence tag approach

De novo sequencinginspect raw data http://qbab.aber.ac.uk

Tandem mass spectra (MS/MS) can be used for peptide sequencing

Mascot Search Results Search title : SampleSetID: 362, AnalysisID: 567, MaldiWellID: 15790, SpectrumID: 17225, Path=\Mani\102004\New Analysis 1 Database : NCBInr 20040606 (1846720 sequences; 611532004 residues) Timestamp : 20 Oct 2004 at 14:52:50 GMT Top Score : 681 for gi|180570, creatine kinase [Homo sapiens]

Probability Based Mowse Score

Score is -10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 75 are significant (p<0.05).

Accession Mass Score Description 1. gi|180570 42591 681 creatine kinase [Homo sapiens] 2. gi|21536286 42617 681 brain creatine kinase; creatine kinase-B [Homo sapiens] 3. gi|33304149 42730 681 creatine kinase, brain [synthetic construct] 4. gi|125292 42674 568 CREATINE KINASE, B CHAIN (B-CK) [Cannis familiaris] 5. gi|180572 42658 538 creatine kinase-B 6. gi|125295 42636 514 CREATINE KINASE, B CHAIN (B-CK) 7. gi|180555 42460 507 creatine kinase-B 8. gi|203476 40598 473 creatine kinase-B 9. gi|31542401 42685 471 creatine kinase, brain [Rattus norvegicus] 10. gi|203474 42699 471 creatine kinase 11. gi|40807002 44540 469 Unknown (protein for IMAGE:5598839) [Rattus norvegicus] 12. gi|47477783 44782 469 Ckb protein [Rattus norvegicus] 13. gi|13096153 42551 441 Chain A, Crystal Structure Of Bovine Retinal Creatine Kinase 14. gi|12852054 42700 427 unnamed protein product [Mus musculus] 15. gi|10946574 42686 427 creatine kinase, brain [Mus musculus] 16. gi|47213348 42953 237 unnamed protein product [Tetraodon nigroviridis] 17. gi|627264 40353 236 creatine kinase (EC 2.7.3.2) isozyme IV - African clawed frog 18. gi|27503418 42214 235 Ckb-prov protein [Xenopus laevis] 19. gi|45384340 42844 209 B-creatine kinase [Gallus gallus] 20. gi|6573489 42713 201 Chain A, Crystal Structure Of Chicken Brain-Type Creatine Kinase

Top hits from Mascot Search – there are multiple accession numbers for the same protein

Search returns a cluster of proteins with the same matching peptides

1. gi|180570 Mass: 42591 Score: 681 creatine kinase [Homo sapiens] Observed Mr(expt) Mr(calc) Delta Start End Miss Ions Peptide 1232.62 1231.61 1231.61 0.00 87 - 96 0 45 DLFDPIIEDR 1232.62 1231.61 1231.61 0.00 87 - 96 0 ---- DLFDPIIEDR 1254.57 1253.56 1253.58 -0.02 97 - 107 0 ---- HGGYKPSDEHK 1303.70 1302.70 1302.72 -0.02 33 - 43 0 ---- VLTPELYAELR 1303.70 1302.70 1302.72 -0.02 33 - 43 0 54 VLTPELYAELR 1458.70 1457.69 1457.67 0.02 139 - 151 1 ---- GFCLPPHCSRGER 1586.81 1585.80 1585.83 -0.03 157 - 172 0 81 LAVEALSSLDGDLAGR 1586.81 1585.80 1585.83 -0.03 157 - 172 0 ---- LAVEALSSLDGDLAGR 1656.79 1655.79 1655.82 -0.03 367 - 381 0 ---- LEQGQAIDDLMPAQK 1657.80 1656.79 1656.83 -0.04 224 - 236 0 47 TFLVWVNEEDHLR 1657.80 1656.79 1656.83 -0.04 224 - 236 0 ---- TFLVWVNEEDHLR 1848.94 1847.93 1847.97 -0.04 342 - 358 0 ---- LGFSEVELVQMVVDGVK 1864.93 1863.92 1863.97 -0.04 342 - 358 0 ---- LGFSEVELVQMVVDGVK 1964.88 1963.88 1963.92 -0.05 321 - 341 0 ---- GTGGVDTAAVGGVFDVSNADR 1964.88 1963.88 1963.92 -0.05 321 - 341 0 139 GTGGVDTAAVGGVFDVSNADR 2120.98 2119.97 2120.02 -0.05 320 - 341 1 ---- RGTGGVDTAAVGGVFDVSNADR 2120.98 2119.97 2120.02 -0.05 320 - 341 1 27 RGTGGVDTAAVGGVFDVSNADR 2169.91 2168.91 2168.96 -0.05 14 - 32 0 ---- FPAEDEFPDLSAHNNHMAK 2225.06 2224.05 2224.17 -0.12 157 - 177 1 ---- LAVEALSSLDGDLAGRYYALK 2439.08 2438.07 2438.14 -0.07 12 - 32 1 31 LRFPAEDEFPDLSAHNNHMAK 2439.08 2438.07 2438.14 -0.07 12 - 32 1 ---- LRFPAEDEFPDLSAHNNHMAK 2518.10 2517.09 2517.16 -0.07 108 - 130 0 92 TDLNPDNLQGGDDLDPNYVLSSR 2518.10 2517.09 2517.16 -0.07 108 - 130 0 ---- TDLNPDNLQGGDDLDPNYVLSSR 3753.61 3752.60 3752.73 -0.13 97 - 130 1 ---- HGGYKPSDEHKTDLNPDNLQGGDDLDPNYVLSSR 3753.61 3752.60 3752.73 -0.13 97 - 130 1 55 HGGYKPSDEHKTDLNPDNLQGGDDLDPNYVLSSR

4. gi|125292 Mass: 42674 Score: 568 CREATINE KINASE, B CHAIN (B-CK) Observed Mr(expt) Mr(calc) Delta Start End Miss Ions Peptide 1254.57 1253.56 1253.58 -0.02 97 - 107 0 ---- HGGYKPSDEHK 1303.70 1302.70 1302.72 -0.02 33 - 43 0 ---- VLTPELYAELR 1303.70 1302.70 1302.72 -0.02 33 - 43 0 54 VLTPELYAELR 1458.70 1457.69 1457.67 0.02 139 - 151 1 ---- GFCLPPHCSRGER 1586.81 1585.80 1585.83 -0.03 157 - 172 0 81 LAVEALSSLDGDLAGR 1586.81 1585.80 1585.83 -0.03 157 - 172 0 ---- LAVEALSSLDGDLAGR 1624.76 1623.75 1623.85 -0.10 367 - 381 0 ---- LEQGQAIDDLVPAQK 1848.94 1847.93 1847.97 -0.04 342 - 358 0 ---- LGFSEVELVQMVVDGVK 1864.93 1863.92 1863.97 -0.04 342 - 358 0 ---- LGFSEVELVQMVVDGVK 1964.88 1963.88 1963.92 -0.05 321 - 341 0 ---- GTGGVDTAAVGGVFDVSNADR 1964.88 1963.88 1963.92 -0.05 321 - 341 0 139 GTGGVDTAAVGGVFDVSNADR 2120.98 2119.97 2120.02 -0.05 320 - 341 1 ---- RGTGGVDTAAVGGVFDVSNADR 2120.98 2119.97 2120.02 -0.05 320 - 341 1 27 RGTGGVDTAAVGGVFDVSNADR 2169.91 2168.91 2168.96 -0.05 14 - 32 0 ---- FPAEDEFPDLSAHNNHMAK 2225.06 2224.05 2224.17 -0.12 157 - 177 1 ---- LAVEALSSLDGDLAGRYYALK 2439.08 2438.07 2438.14 -0.07 12 - 32 1 31 LRFPAEDEFPDLSAHNNHMAK 2439.08 2438.07 2438.14 -0.07 12 - 32 1 ---- LRFPAEDEFPDLSAHNNHMAK 2518.10 2517.09 2517.16 -0.07 108 - 130 0 92 TDLNPDNLQGGDDLDPNYVLSSR 2518.10 2517.09 2517.16 -0.07 108 - 130 0 ---- TDLNPDNLQGGDDLDPNYVLSSR 3753.61 3752.60 3752.73 -0.13 97 - 130 1 ---- HGGYKPSDEHKTDLNPDNLQGGDDLDPNYVLSSR 3753.61 3752.60 3752.73 -0.13 97 - 130 1 55 HGGYKPSDEHKTDLNPDNLQGGDDLDPNYVLSSR

Nominal mass (Mr): 42591; Calculated pI value: 5.34Observed Mass & pI: 43kd, 6.2-6.27

Creatine kinase - B [Homo sapiens]Match to: gi|21536286 ; Score: 681

Sequence Coverage: 46%

1 MPFSNSHNAL KLRFPAEDEF PDLSAHNNHM AKVLTPELYA ELRAKSTPSG

51 FTLDDVIQTG VDNPGHPYIM TVGCVAGDEE SYEVFKDLFD PIIEDRHGGY

101 KPSDEHKTDL NPDNLQGGDD LDPNYVLSSR VRTGRSIRGF CLPPHCSRGE

151 RRAIEKLAVE ALSSLDGDLA GRYYALKSMT EAEQQQLIDD HFLFDKPVSP

201 LLSASGMARD WPDARGIWHN DNKTFLVWVN EEDHLRVISM QKGGNMKEVF

251 TRFCTGLTQI ETLFKSKDYE FMWNPHLGYI LTCPSNLGTG LRAGVHIKLP

301 NLGKHEKFSE VLKRLRLQKR GTGGVDTAAV GGVFDVSNAD RLGFSEVELV

351 QMVVDGVKLL IEMEQRLEQG QAIDDLMPAQ K

Creatine kinase B is the highest scoring protein

outline

Mass spectrometers Protein identification Quantitative proteomics Protein-protein interactions

Sample preparation

Quantitative Proteomics

From 2D gels ….to MALDI or ESI MS

Control Test

Cy3 Cy5

Pool

Image analysis with Delta2D, DecodonQuantitate

Export spot list to robotic picker

2nd Dimension – SDS PAGE

1st Dimension - Isoelectric focussing

Spot picking

Trypsin gel digest

..its high-throughput…

Colorectal cancer markersMass spectral analysis

MS

MS/MSm/z

m/z

Database Search

Protein Identified

YesImmunohistochemistry

2D

1D

Immunoblotting Validation

Isolate Nuclear Matrix

In-gelTryptic digest

No

Tumor specific markersCC3, CC4, CC5, CC6a, CC6b

de novo sequencing

Balasubramani et al., Cancer Res., 2006

Shotgun proteomics

Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207

…typical workflow to identify biomarkers that distinguish indolent versus aggressive forms of cancer..

Group A, Indolent Group B, Aggressive

FractionateEg. Immunodeplete, subcellular

FractionateEg. Immunodeplete, subcellular

Tryptic peptides Tryptic peptides

Label with iTRAQ reagent 115 Label with iTRAQ reagent 116

Combine labeled digestsLC fractionate

MS and MS/MS

Protein ID and Quantitate

Sample handling

HPLC

1D or 2D LC MALDIIn-solutionIsoelectricfocussing

Protein-protein interaction studies Immunoaffinity pull-downs Tandem affinity purification

GPCL Billy W DayPaul Wood

Mirunalni ThangaveluTamanna SultanaEmanuel M SchreiberChris BolcatoChris Myers

Patrick MillerRobert Wolfe

definitions The amu is defined as 1/12th the mass of

one neutral 6C12 atom Amu is also called the dalton 1 amu =1/12 ( 12g 12C/mol 12C

6.0221 x 1023 atoms 12C/mol 12C

1.6605 x 10-24 g/atom 12C

Isotopic species of M (M + H)+ (M + 1H)/1H+

(M + 2H)2+ (M + 2H)/2H+

(M + 3H)3+ (M + 3H)/3H+