Wikiomics:Protein mass spectrometry

Protein mass spectrometry can be divided into:

• identification of proteins/peptides
• quantification

Protein/peptide identification

Peptide Mass Fingerprinting (PMF) or (MS)

Old method, superseded by MS/MS

• algorithms:

• • Mascot (gives probabilistic score)
  • Aldente
  • ProFound ProFound

• caveats
  • no sequence information
  • journals started to require that at least one peptide of a protein identified by PMF should be confirmed by MS/MS

Peptide fragment fingerprinting (PFF) or (MS/MS)

• algorithms (most commonly used):
  • Sequest $$$
  • Mascot $$$
  • OMSSA Open Mass Spectrometry Search Algorithm, open source
  • XTandem open source effort from Canada

• algorithms (other/new/experimental):
  • Spectrum Mill $$$
  • MASPIC
    • this paper claims 5-15% more confident hits than Sequest: [1]
  • InsPecT A new variable mods search from Pevzner & Tanner @UCSD (free?)

• filtering of the results
  • Trans Proteomic Pipeline [2] (free?)
    • download from Sourceforge (TPP Cygwin Setup for Windows or 'Trans-Proteomic Pipeline' for Linux)
    • commercial offshot IPP
    • wiki devoted to TPP TPP_Wiki
    • dynamic newsgroup: spctools-discuss

• • DTASelect it seems to be in a semi-frozen state (free for nonprofit but requires signed MTA)

Databases

Protein databases

Use (if possible):

• IPI International Protein Index
• always use target-decoy database (i.e. concatenated: human_IPI + reversed_human_IPI)
• decoy databases creation methods:
  • protein reversal (simple to perform. does not scramble fortunately quite rare palindromic sequences)
    • MEGGAYGAGKAGGAFDPYTL -=> LTYPDFAGGAKGAGYAGGEM
  • peptide pseudo-reversal (used in Sorcerer by Sage-N Research)
    • MEGGAYGAGKAGGAFDPYTL => (trypsin digest, used Ms-Digest) MEGGAYGAGK AGGAFDPYTL => GAGYAGGEMK-ALTYPDFAGG (each peptide reversed, but trypsin digestion site preserved -> guessed from the Elias 2007 paper)
  • shuffled
    • MEGGAYGAGKAGGAFDPYTL => FYAGADEAGMGTYKGGAGLP (used SMS, results differ each time) -> recommended by EBI ppl
  • random (i.e. creating database of random proteins based on frequency of AA in source fasta file)
• to create decoy database use DBToolkit free java standalone

Modification databases

• Unimod (> 500 natural + labels)
• Delta Mass A Database of Protein Post Translational Modifications (in vivo)
• RESID detailed descriptions of > 400 modifications

Peptide Tag Searching

• GutenTag free for non-profit, MTA required. Assigns fewer peptides than Sequest but with fewer false positives. Occupies a middle ground between mainstream search algorithms and de novo sequencing.

de novo sequence determination algorithms

• PepNovo: (PDF)
• Sherenga (PDF)
• Peaks (PDF)
• Lutefisk web

Spectral matching

The idea is that if one can match spectrum of an unknown peptide to a very similar MS/MS spectrum in a database with a determined sequence/annotation then one can annotate unknown peptide in a process similar to orthologue annotation in protein sequence databases. Caveat: bad annotations will also get propagated.

• P3 (server) from Global Proteomics Machine (free)
  • description
• SpectraST from ISB, Seattle (not as many species/options as P3)
• BiblioSpec from MacCoss lab. (free for non-profit, online licence)
  • command line only

Protein quantification

• approaches
  • isotopic labeling (ICAT, ITRAQ, SILAC, 18O- or 15N-labeling)
  • label-free methods

• software
  • ASAPRatio from Trans Proteomics Pipeline:

"calculates the relative abundances of proteins and the corresponding confidence intervals from ICAT-type ESI-LC/MS data"

• • MSQuant Parser for Mascot results for quantitation (Windows only)

Frameworks/pipelines

• Trans Proteomic Pipeline (TPP) most popular, included in Sorcerer from Sage-N Research. Windows/Cygwin/Perl or Linux based.
• Open-MS German, C++ based
• Sorcerer $$$ FPGA-based fast hardware solution for SEQUEST & Tandem searches with TPP on top of it.

Web sites

• UCSD (Pevzner)
• U. of Washington (MacCoss)
• Proteome Commons collection of tools & links
• GenePattern proteomics modules from Broad Inst.

Reviews

For a good review of programs and aspects of protein identification by mass spectrometry see:

• Hernandez et al. 2006 (HTML)

• Palagi et al. 2006 (PDF)

• Shadforth et al. 2005 (PDF)

Other tools to be sorted out

• ProteinLynx Global SERVER $$$, from Waters Waters Corporation

• massSorter
• ProteinProspector
• Sonar

• DeNovoID web
• SPIDER (PDF) de novo + homology search in other species
• OpenSea (HTML) Java program available from authors

• ModifiComb (HTML) (available from authors?)
• MODi web server for PTMs discovery

• SILVER view your spectra with LOD scores

Credits

• Darek Kedra wrote this tutorial