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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M800472-MCP200v1 8/8/2011    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Volchenboum, S. L. Articles by Kron, S. J. Search for Related Content PubMed PubMed Citation Articles by Volchenboum, S. L. Articles by Kron, S. J. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 8:2011-2022, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Rapid Validation of Mascot Search Results via Stable Isotope Labeling, Pair Picking, and Deconvolution of Fragmentation Patterns^*,

Samuel L. Volchenboum^,,¶, Kolbrun Kristjansdottir^||,**, Donald Wolfgeher^** and Stephen J. Kron^||,**

From the Departments of Pediatrics and
||Molecular Genetics and Cell Biology,
Computation Institute, and
**Ludwig Center for Metastasis Research, The University of Chicago, Chicago, Illinois 60637

Conventional LC-MS/MS data analysis matches each precursor ion and fragmentation pattern to their best fit within databases of theoretical spectra, yielding a peptide identification. Confidence is estimated by a score but can be validated by statistics, false discovery rates, and/or manual validation. A weakness is that each ion is evaluated independently, discarding potentially useful cross-correlations. In a classical approach to de novo sequence analysis, mixtures of peptides differing only in a carboxyl-terminal isotopic label yield fragmentation spectra with single, unlabeled b-type ions but pairs of isotope-labeled y-type ions, facilitating confident assignments. To apply this principle to identification by fragmentation pattern matching, we developed Validator, software that recognizes isotopic peptide pairs and compares their identifications and fragmentation patterns. Testing Validator 1 on a Mascot results file from FT-ICR LC-MS/MS of ¹⁶O/¹⁸O-labeled yeast cell lysate peptides yielded 2,775 peptide pairs sharing a common identification but differing in carboxyl-terminal label. Comparing observed b- and y-ions with the predicted fragmentation pattern improved the threshold Mascot score for 5% false discovery from 36 to 22, significantly increasing both sensitivity and specificity. Validator 2, which identifies pairs by precursor mass difference alone before comparing observed fragmentation with that predicted by Mascot, found 2,021 isotopic pairs, similarly achieving improved sensitivity and specificity. Finally Validator 3, which finds pairs based on mass difference alone and then deconvolutes fragmentation patterns independently of Mascot, found 964 predicted peptides. Validator 3 allowed raw mass spectrometry data to be mined not only to validate Mascot results but also to discover peptides missed by Mascot. Using standard desktop hardware, the Validator 1–3 software processed the 11,536 spectra in the 93-MB Mascot .DAT file in less than 6 min (32 spectra/s), revealing high confidence peptide identifications without regard to Mascot score, far faster than manual or other independent validation methods.

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