introduction to mass spectrometry-based protein identification and quantification
DESCRIPTION
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 R - PowerPoint PPT PresentationTRANSCRIPT
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.
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
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
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: [email protected]; 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: [email protected] Phone number: 8-2031