Introduction to mass spectrometry-based protein identification and quantification

Austin Yang, Ph.D.

Aebersold R, Mann M.Mass spectrometry-based proteomics.Nature. 2003 Mar 13;422(6928):198-207. Review.

Mueller LN, Brusniak MY, Mani DR, Aebersold RAn assessment of software solutions for the analysis of mass spectrometry based quantitative proteomics data.J Proteome Res. 2008 Jan;7(1):51-61.

The typical proteomics experiment consists of five stages

Mass spectrometers used in proteome research.

Monoistopic Mass = 1155.6

Average Mass = 1156.3 (calculated)

As shown in Figure 1. the monoisotoptic mass of this compound is 1155.6. For a given compound the monoisotopic mass is the mass of the isotopic peak whose elemental composition is composed of the most abundant isotopes of those elements. The monoisotopic mass can be calculated using the atomic masses of the isotopes.

The average mass is the weighted average of the isotopic masses weighted by the isotopic abundances. The average mass can be calculated using the atomic weights of the elements.

www.ionsource.com

Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes

Iso

A %Iso

A+1 %Iso

A+2 %Iso

A+3 %Iso

A+4 %

12C

12 98.93(8)13C

13.0033548378(10) 1.07(8)14C

14.003241988(4) - - - - - - -

1H 1.0078250321(4) 99.9885(70)2H

2.0141017780(4)0.0115(

70)3H 3.0160492675(11) - - - - - - -

14N

14.0030740052(9) 99.632(7)15N

15.0001088984(9) 0.368(7) - - - - - - - - -

16O

15.9949146221(15) 99.757(16)17O

16.99913150(22) 0.038(1)18O

17.9991604(9) 0.205(14) - - - - - -

32S 31.97207069(12) 94.93(31)33S

32.97145850(12) 0.76(2)34S

33.96786683(11) 4.29(28) - - -36

S35.96708088(25) 0.02(1)

. . . . . . . . . . . . . . .

19F 18.99840320(7) 100 - - - - - - - - - - - -

23Na

22.98976967(23) 100 - - - - - - - - - - - -

39K

38.9637069(3) 93.2581(44)40K

39.96399867(29)0.0117(

1)41K

40.96182597(28)6.7302(44

)- - - - - -

31P 30.97376151(20) 100 - - - - - - - - - - - -

35Cl

34.96885271(4) 75.781(4) - - -37Cl

36.96590260(5) 24.22(4) - - - - - -

55Mn

54.9380496(14) 100 - - - - - - - - - - - -

54Fe

53.9396148(14) 5.845(35) - - -56Fe

55.9349421(15)91.754(36

)

57Fe

56.9353987(15)2.119(10)

58

Fe57.9332805(15)

0.282(4)

63Cu

62.9296011(15) 69.17(3) - - -65Cu

64.9277937(19) 30.83(3) - - - - - -

79Br

78.9183376(20) 50.69(7) - - -81Br

80.916291(3) 49.31(7) - - - - - -

127I

126.904468(4) 100 - - - - - - - - - - - -

Peak Abundance, “Mass Crossover” and Calibration

The Nobel Prize in Chemistry 2002The Nobel Prize in Chemistry 2002

"for the development of methods for identification and "for the development of methods for identification and structure analyses of biological macromolecules" structure analyses of biological macromolecules"

"for their development of soft desorption ionisation "for their development of soft desorption ionisation methods for mass spectrometric analyses of biological methods for mass spectrometric analyses of biological macromolecules"macromolecules"

John FennJohn Fenn Koichi TanakaKoichi Tanaka

Mass Spectrometry:Mass Spectrometry:A method to “weigh” moleculesA method to “weigh” molecules

A simple measurement of A simple measurement of mass is used to confirm the mass is used to confirm the identity of a molecule, but it identity of a molecule, but it can be used for much can be used for much more……more……

Other information can be inferred Other information can be inferred from a weight measurement.from a weight measurement.

• Post-translational modificationsPost-translational modifications• Molecular interactionsMolecular interactions• ShapeShape• SequenceSequence• Physical dimensionsPhysical dimensions• etc...etc...

high voltagehigh voltage

samplesample

vv11

m1

vv22

m2

vv33

m3

m1 m2 m3

detectordetector

drift regionlaserlaser

Matrix-assisted Laser Matrix-assisted Laser Desorption/Ionization (MALDI)Desorption/Ionization (MALDI)

Time-of-Flight (TOF) AnalyzerTime-of-Flight (TOF) Analyzer

MALDIMALDI

Electrospray: Generation of aerosols and droplets

“Wings to Molecular Elephants”

Electrospray Ionization (ESI)Electrospray Ionization (ESI)

• Multiple chargingMultiple charging– More charges for larger moleculesMore charges for larger molecules

• MW range > 150 kDaMW range > 150 kDa• Liquid introduction of analyteLiquid introduction of analyte

– Interface with liquid separation Interface with liquid separation methods, e.g. liquid methods, e.g. liquid chromatographychromatography

– Tandem mass spectrometry Tandem mass spectrometry (MS/MS) for protein sequencing(MS/MS) for protein sequencing

500500 700700 900900 11001100

mass/charge (mass/charge (m/zm/z))

20+20+19+19+

18+18+

17+17+

16+16+

15+15+14+14+

21+21+

22+22+

highly charge highly charge dropletsdroplets

MSMS

ESIESI

Origin of the ES Spectra of PeptidesOrigin of the ES Spectra of Peptides

H

H

HH

4+

HH

H

3+

H

2+

H

1+

H

m/z = (Mr+4H)/4

m/z = (Mr+3H)/3

m/z = (Mr+2H)/2

m/z = (Mr+H)

1+

2+

3+

4+

Rel. Inten.

m/z

ES-MS

b1

b2

b3

y1

y2

y3

LF G K

Rela

t ive I

nte

nsit

y

m/z

F L G K

++

F L G K

++

F L G K

++

CID

F L G K++

F L G K

++

F L G K

++

b1

b2

b3

y3

y2

y1 F L G K

++

F L G K

+

Theoretical CID of a Tryptic Peptide

K G L F

MS/MSSpectrum

Parentions

(464.29)

Daughter ionsNon-dissociatedParent ions

Peptide Sequencing by LC/MS/MS

Web addresses of some representative internet resources for protein identification from mass

spectrometry data

Program Web Address

BLAST http:/ / www.ebi.ac.uk/ blastall/

Mascot http:/ / www.matrixscience.com/ cgi/ index.pl?page=/ home.html

MassSearch http:/ / cbrg.inf .ethz.ch/ Server/ ServerBooklet/ MassSearchEx.html

MOWSE http:/ / srs.hgmp.mrc.ac.uk/ cgi-bin/ mowse

PeptideSearch http:/ / www.narrador.embl-

heidelberg.de/ GroupPages/ PageLink/ peptidesearchpage.html

Protein Prospector http:/ / prospector.ucsf .edu/

Prowl http:/ / prowl.rockefeller.edu/

SEQUEST http:/ / fields.scripps.edu/ sequest/

Data Mining through SEQUEST and PAULA

Database Search Time•Yeast ORFs (6,351 entries) 52 sec: 0.104 sec/s•Non-redundant protein (100k entries) 3500 min: •EST (100K entries, 3-frames) 5-10,000 min:

SEQ 1

SEQ 2

SEQ 3

SEQ 4

STEP 1.

STEP 3.

SEQUEST Algorithm

(Experimental MS/MS Spectrum)

500 peptides with masses closest to that of the parent ion are retrieved from a protein database. Computer generates a theoretical MS/MS Spectrum for each peptide sequence (SEQ1, 2, 3, 4, …)

(Experimental MS/MS Spectrum)

Theoretical MS/MSspectra

Step 1.Determine Parent

Ion molecular mass

Step 2.

Step 3.Experimental Spectrum is compared with each theoretical spectra and correlation scores are assigned.

Step 4.Scores are ranked andProtein Identifications are made based on these cross correlation scores.

ZSA-charge assignment

Unified Scoring Function

Prot APeptide 1

Peptide 2

Prot BPeptide 3

Peptide 4

Peptide 5

Prot

Prot

Prot

Prot

in the sample(enriched for ‘multi-hit’ proteins)

not in the sample(enriched for ‘single hits’)Prot

Peptide 6

Peptide 7

Peptide 8

Peptide 9

Peptide10

+

++

+

+

5correct (+)

Amplification of False Positive Error Rate from Peptide to Protein Level

Peptide Level: 50% False Positives

Protein Level: 71% False Positives

Quantitative Mass Spec Analysis

1. Relative Quantitationa. SILAC and iTRAQb. Digestion with Oxygen-18 Waterc. Spectra Counting and Non-labeling Methodology 2. Absolute Quantitation

Trypsin Digestion with Oxygen18 and Oxygen16 Water

Limitation of SILAC

Philip L. Ross, et al. Molecular & Cellular Proteomics 3:1154–1169, 2004.

Multiplexed Isobaric Tagging Technology (iTRAQ)

Isobaric Tag = 145

BSA114_115_21 #6272 RT: 33.88 AV: 1 NL: 1.99E6T: ITMS + c ESI d Full ms2 650.54@pqd21.00 [50.00-1965.00]

200 400 600 800 1000 1200 1400 1600 1800m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative A

bundance

650.29

649.67

928.15371.76 653.27619.71 1437.901054.49 1154.70 1560.73 1647.55133.01

BSA114_117_31 #6446 RT: 34.78 AV: 1 NL: 1.58E5T: ITMS + c ESI d Full ms2 650.32@pqd31.00 [50.00-1965.00]

200 400 600 800 1000 1200 1400 1600 1800m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tiv

e A

bundance

371.56

372.42

927.89

929.26

420.28 742.55578.07822.79291.36 635.59

145.131010.58 1435.841154.58 1251.79 1536.66 1711.79

BSA114_117_35 #6127 RT: 32.86 AV: 1 NL: 1.34E4T: ITMS + c ESI d Full ms2 650.28@pqd35.00 [50.00-1965.00]

200 400 600 800 1000 1200 1400 1600 1800m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tiv

e A

bundance

928.57

420.15

742.65

291.14402.30

606.37114.05 735.56

878.72578.14

772.20145.06

228.20 1010.13 1430.54

1501.851034.501167.64

1566.86

Release of 114 and 117 Reporter Ions

Parent Ion

Regular CID to obtain sequenceLow mass cut-off and no reporter ion

High Energy Collision Cellto quantify and sequence

PSD_117: PSD_114=2:1Loading 10ug9 salt cuts online 2D_LC_MS/MS962 proteins are quantified

Expected ratio

Protein name117/114

ratioNum of

pep PSD93 2.829 5PSD95 2.021 21PSD95-AP1 1.764 2GABA alpha 1.365 2GABA beta 2.087 3NR2B 1.813 4AMPA1 2.092 7AMPA2 1.921 11AMPA4 1.902 4NR1 1.658 6

Johri et al. Nature Reviews Microbiology 4, 932 – 942 (December 2006) | doi:10.1038/ nrmicro1552

Absolute Quantification

Public Web Serverhttp://www.matrixscience.com/search_form_select.html

Class Data Download:http://10.90.157.112/GPLS716

Local Web Serverhttp://10.90.157.112/mascotUsername: GPILSPassword: GPILS

MS1 PMF(peptide mass fingerprinting) Search Example

• Data: testms1.txt, 210 MS1 peaks• Database: bovine• Fixed modifications : Carboxymethyl (C)

Variable modifications : Oxidation (M)• Peptide Tolerance: 0.1 Da• Monoisotopic mass• Mass Value: Mr

Quantification Search Example• Note: Save link as; Save this file to the desktop)• Data:

18O_BSA_100fmol_1to5_01_071018.RAW.mgf• Database: bovine• Fixed modifications : Carbamidomethyl (C)• Peptide Tolerance: 8 Da (required for O18 labeling)• Fragment Tolerance: 0.2 Da• Peptide Charge: Mr• Quantification Method: 18O corrected multiplex

MS/MS Database Search Example

• Data: BSA onespectra.mgf (one spectra)• Database: bovine• Fixed modifications: Carboxymethyl(C + 58.01)• Varied modifications: Oxidatation(M)• Peptide Mass Tolerance : 0.1 Da • Fragment Mass Tolerance: 0.1 Da • http://www.matrixscience.com/help/

fragmentation_help.html

Cysteine C3H5NOS 103.00918

Carboxymethyl Cys C5H7NO3S 161.01466 58.00548

Alkylation of Cysteine Residue

MS2 mixture example

• Data: mixture10spectra.mgf• Database: yeast• Fixed modifications : Carbamidomethyl (C+57.02) • Variable modifications : Oxidation (M)• Peptide Mass Tolerance : 0.1 Da • Fragment Mass Tolerance: 0.1 Da

Home Work1. You will have to download your datasets from the following url:http://10.90.157.112/GPLS716 a. Identification of phosphorylation site : Data:BIG3021307.RAW.mgf Recommend parameters: Database: human. Variable Modification: Phospho(ST) Fixed modification: Carboamidomethyl(C).

b. Quantificaiton of oxygen-18/oxygen-16 digested BSA

Data: 18O_BSA_500fmol_071013.RAW.mgf.

Submit your search results in pdf or html format to the following email address: proteomicsumb@gmail.com; Please include the following information when you submit your homework

1. Your name and ID in the subject of your email 2. Search parameters

3. A short summary of your search results.

Questions: Contact Yunhu Wan, email: ywan@som.umaryland.edu Phone number: 8-2031