Proteome Bioinformatics - Vanderbilt...
Transcript of Proteome Bioinformatics - Vanderbilt...
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Proteome Bioinformatics
Applied Bioinformatics lecture 6
David L. Tabb
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Overview
• Identifying proteins through “shotgun”
proteomics: bench and bioinformatics
• Understanding Peptide-Spectrum Matches
• Trawling ProteomExchange via PeptideAtlas
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Mass spectrometers
• IONIZATION:– Produce ions from biological materials.
• MASS ANALYSIS:– Separate or select ions by mass-to-charge (m/z)
ratio.
• DETECTION:– Report intensity of ions in mass spectrum.
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Discovery Proteomics
Peptide
Mixture
Liquid
Chromatography
Electrospray
Ionization
High-Resolution
Mass Spectrometry
Isolate
Ions of Peptide
Collide Ions to
Dissociate
Collect Fragments
in Tandem MS
Tandem
Mass spectra
Peptide
Identifications
Confident
Peptide List
Assembled
Protein List
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Disassembly and reassembly
Collection of tandem
mass spectra
Collection of raw
peptide identifications
LSELIGAR
z=2 XCorr=3.5
Mixture of PeptidesConfidently identified
peptide sequences
...LSEGTSFR
LSELIGAR
LSENLRK
LSEPVHK...
Mixture of Proteins Confidently identified
proteins
...YGR192C
YGR204W
YGR208W
YGR209C...
After AI Nesvizhskii, Mol Cell Proteomics (2005) 4: 1419-40.
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Database search algorithms
First published in 1994, these tools identify MS/MS scans by comparing them
to predictions from database peptide sequences. Prominent examples include:
Sequest : Eng (1994) J. Amer. Soc. Mass Spectrom. 5: 976-989.
Mascot: Perkins (1999) Electrophoresis 20: 3551-3567.
X!Tandem: Craig (2003) Rapid Comm. Mass Spectrom. 17: 2310-2316.
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Fragment ions result from breakage of peptide bonds
TSIIGTIGPK
N-terminal
b ionC-terminal
y ion
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HFISELEK, +2 charge state
HF-
-LEK -SELEK
-ISELEK
-FISELEK
Neutral loss of
water from peptide
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Proteomic Repositories
Web resources of proteomic data have become substantial in recent years.
Raw data and
Identification archives
Peer-to-peer file storage
Data analysis tools
and databases
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Peptide Atlas: protein coverage
http://www.peptideatlas.org search for NP_862897 in mouse build
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Peptide-spectrum match view
predicted and observed
fragment ions
y ion contains
peptide C-terminusb ion contains
peptide N-terminus
Distance between
b8 and b9 is mass
of ninth amino acid Tabb (2006) Nat. Protocols 1: 2213-2222.
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Summary
• Proteomics generates large data sets that require automated interpretation.
• Proteomic repositories are relatively new, and interpreting experiments requires expertise.
• Visual inspection of tandem mass spectra is largely built from rules-of-thumb.
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Challenges
• What areas of mouse dihydropyrimidinedehydrogenase (NP_740748.1) have been observed by proteomics?
• I want to create a targeted measurement of mouse adenylyl cyclase-associated protein 1 (NP_031624.2). What charge state does KEPALLELEGK adopt?