Improving Reproducibility and Sensitivity in Identifying Human Proteins by Shotgun Proteomics

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• 作者：Katheryn A. Resing ; Karen Meyer-Arendt ; Alex M. Mendoza ; Lauren D. Aveline-Wolf ; Karen R. Jonscher ; Kevin G. Pierce ; William M. Old ; Hiu T. Cheung ; Steven Russell ; Joy L. Wattawa ; Geoff R. Goehle ; Rob
• 刊名：Analytical Chemistry
• 出版年：2004
• 出版时间：July 1, 2004
• 年：2004
• 卷：76
• 期：13
• 页码：3556 - 3568
• 全文大小：203K
• 年卷期：v.76,no.13(July 1, 2004)
• ISSN：1520-6882

文摘

Identifying proteins in cell extracts by shotgun proteomicsinvolves digesting the proteins, sequencing the resultingpeptides by data-dependent mass spectrometry (MS/MS),and searching protein databases to identify the proteinsfrom which the peptides are derived. Manual analysis anddirect spectral comparison reveal that scores from twocommonly used search programs (Sequest and Mascot)validate less than half of potentially identifiable MS/MSspectra (class positive) from shotgun analyses of thehuman erythroleukemia K562 cell line. Here we demonstrate increased sensitivity and accuracy using a focusedsearch strategy along with a peptide sequence validationscript that does not rely exclusively on XCorr or Mowsescores generated by Sequest or Mascot, but uses consensus between the search programs, along with chemicalproperties and scores describing the nature of the fragmentation spectrum (ion score and RSP). The approachyielded 4.2% false positive and 8% false negative frequencies in peptide assignments. The protein profile is thenassembled from peptide assignments using a novel peptide-centric protein nomenclature that more accuratelyreports protein variants that contain identical peptidesequences. An Isoform Resolver algorithm ensures thatthe protein count is not inflated by variants in the proteindatabase, eliminating ~25% of redundant proteins. Analysis of soluble proteins from a human K562 cells identified5130 unique proteins, with ~100 false positive proteinassignments.