Integrating "top-down" and "bottom-up" mass spectrometric approaches for proteomic analysis of Shewanella oneidensis

Nathan C. VerBerkmoes, Jonathan L. Bundy, Loren Hauser, Keiji G. Asano, Jane Razumovskaya, Frank Larimer, Robert L. Hettich, James L. Stephenson

Research output: Contribution to journalArticlepeer-review

125 Scopus citations

Abstract

Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by online liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This "top-down" technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the "bottom-up" experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism.

Original languageEnglish
Pages (from-to)239-252
Number of pages14
JournalJournal of Proteome Research
Volume1
Issue number3
DOIs
StatePublished - May 2002

Keywords

  • Database searching
  • Intact proteins
  • LC-MS/MS
  • Mass spectrometry
  • Microbial proteomics

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