Processing complex mixtures of intact proteins for direct analysis by mass spectrometry

Fanyu Meng, Benjamin J. Cargile, Steven M. Patrie, Jeffrey R. Johnson, Shaun M. McLoughlin, Neil L. Kelleher

Research output: Contribution to journalArticlepeer-review

151 Scopus citations

Abstract

For analysis of intact proteins by mass spectrometry (MS), a new twist to a two-dimensional approach to proteome fractionation employs an acid-labile detergent instead of sodium dodecyl sulfate during continuous-elution gel electrophoresis. Use of this acid-labile surfactant (ALS) facilitates subsequent reversed-phase liquid chromatography (RPLC) for a net two-dimensional fractionation illustrated by transforming thousands of intact proteins from Saccharomyces cerevisiae to mixtures of 5-20 components (all within ∼5 kDa of one another) for presentation via electrospray ionization (ESI) to a Fourier transform MS (FTMS). Between 3 and 13 proteins have been detected directly using ESI-FTMS (or MALDI-TOF), and the fractionation showed a peak capacity of ∼400 between 0 and 70 kDa. A probability-based identification was made automatically from raw MS/MS data (obtained using a quadrupole-FTMS hybrid instrument) for one protein that differed from that predicted in a yeast database of ∼19 000 protein forms. This ALS-PAGE/RPLC approach to proteome processing ameliorates the "front end" problem that accompanies direct analysis of whole proteins and assists the future realization of protein identification with 100% sequence coverage in a high-throughput format.

Original languageEnglish (US)
Pages (from-to)2923-2929
Number of pages7
JournalAnalytical Chemistry
Volume74
Issue number13
DOIs
StatePublished - Jul 1 2002

ASJC Scopus subject areas

  • Analytical Chemistry

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