Web and database software for identification of intact proteins using "top down" mass spectrometry

Gregory K. Taylor, Yong Bin Kim, Andrew J. Forbes, Fanyu Meng, Ryan McCarthy, Neil L. Kelleher*

*Corresponding author for this work

Research output: Contribution to journalArticle

122 Scopus citations

Abstract

For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da Δm on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp 90 and Asp 192 , consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/).

Original languageEnglish (US)
Pages (from-to)4081-4086
Number of pages6
JournalAnalytical Chemistry
Volume75
Issue number16
DOIs
StatePublished - Aug 15 2003

ASJC Scopus subject areas

  • Analytical Chemistry

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