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 journalArticle

26 Citations (Scopus)

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 1 2007

Fingerprint

Isoelectric Focusing
Complex Mixtures
Lysine
Post Translational Protein Processing
Peptides
Isoelectric Point
Databases
Acetylation
Protein Databases
Tandem Mass Spectrometry
Electrophoresis
Proteomics
Yeast
Mass spectrometry
Yeasts
Amino Acids
Proteins

Keywords

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

Cite this

Preparative peptide isoelectric focusing as a tool for improving the identification of lysine-acetylated peptides from complex mixtures. / Xie, Hongwei; Bandhakavi, Sricharan; Roe, Mikel R.; Griffin, Timothy J.

In: Journal of Proteome Research, Vol. 6, No. 5, 01.05.2007, p. 2019-2026.

Research output: Contribution to journalArticle

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AU - Bandhakavi, Sricharan

AU - Roe, Mikel R.

AU - Griffin, Timothy J.

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N2 - 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.

AB - 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.

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