Preparative peptide isoelectric focusing as a tool for improving the identification of lysine-acetylated peptides from complex mixtures

Hongwei Xie, Sricharan Bandhakavi, Mikel R. Roe, Timothy J. Griffin

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

Protein sequence database searching of tandem mass spectrometry data is commonly employed to identify post-translational modifications (PTMs) to peptides in global proteomic studies. In these studies, the accurate identification of these modified peptides relies on strategies to ensure high-confidence results from sequence database searching in which differential mass shift parameters are employed to identify PTMs to specific amino acids. Using lysine acetylation as an example PTM, we have observed that the inclusion of differential modification information in sequence database searching dramatically increases the potential for false-positive sequence matches to modified peptides, making the confident identification of true sequence matches difficult. In a proof-of-principle study of whole cell yeast lysates, we demonstrate the combination of preparative isoelectric focusing using free-flow electrophoresis, and an adjusted peptide isoelectric point prediction algorithm, as an effective means to increase the confidence of lysine-acetylated peptide identification. These results demonstrate the potential utility of this general strategy for improving the identification of PTMs which cause a shift to the intrinsic isoelectric point of peptides.

Original languageEnglish (US)
Pages (from-to)2019-2026
Number of pages8
JournalJournal of Proteome Research
Volume6
Issue number5
DOIs
StatePublished - May 2007

Keywords

  • Acetylation
  • False-positive sequence match
  • Free-flow electrophoresis
  • Peptide isoelectric focusing
  • Post-translational modification
  • Proteomic
  • Tandem mass spectrometry

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