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

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. ICAT: Isotope-Coded Affinity Tagsb. Digestion with Oxygen-18 Waterc. Spectra Counting and Non-labeling Methodology 2. Absolute Quantitation

Cysteine C3H5NOS 103.00918

Carboxymethyl Cys C5H7NO3S 161.01466 58.00548

Alkylation of Cysteine Residue

Mascot Example Slides

ICAT

Trypsin Digestion with Oxygen18 and Oxygen16 Water

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://134.192.153.220/GPLS716

Local Web Serverhttp://134.192.153.220/mascotUsername: GPLS716Password: GPLS716

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

• 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• 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

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://134.192.153.220/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_1to5_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