Coupling protein complex analysis to peptide based proteomics

Qiang Gao, Ashraf G. Madian, Xiuping Liu, Jiri Adamec, Fred E. Regnier

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

Proteolysis is a central component of most proteomics methods. Unfortunately much of the information relating to the structural diversity of proteins is lost during digestion. This paper describes a method in which the native proteome of yeast was subjected to preliminary fractionation by size exclusion chromatography (SEC) prior to trypsin digestion of SEC fractions and reversed phase chromatography-mass spectral analysis to identify tryptic peptides thus generated. Through this approach proteins associated with other proteins in high molecular mass complexes were recognized and identified. A focus of this work was on the identification of Hub proteins that associate with multiple interaction partners. A critical component of this strategy is to choose methods and conditions that maximize retention of native structure during the various stages of analysis prior to proteolysis, especially during cell lysis. Maximum survival of protein complexes during lysis was obtained with the French press and bead-beater methods of cell disruption at approximately pH 8 with 200. mM NaCl in the lysis buffer. Structure retention was favored by higher ionic strength, suggesting that hydrophobic effects are important in maintaining the structure of protein complexes. Recovery of protein complexes declined substantially with storage at any temperature, but storage at -20 °C was best when low temperature storage was necessary. Slightly lower recovery was obtained with storage at -80 °C while lowest recovery was achieved at 4 °C. It was concluded that initial fractionation of native proteins in cell lysates by SEC prior to RPC-MS/MS of tryptic digests can be used to recognize and identify proteins in complexes along with their interaction partners in known protein complexes.

Original languageEnglish (US)
Pages (from-to)7661-7668
Number of pages8
JournalJournal of Chromatography A
Volume1217
Issue number49
DOIs
StatePublished - Dec 3 2010

Keywords

  • Hub proteins
  • Protein complex
  • Proteomics
  • Size-exclusion chromatography

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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