Direct ampholyte-free liquid-phase isoelectric peptide focusing: Application to the human serum proteome

Zhen Xiao, Thomas P. Conrads, David A. Lucas, George M. Janini, Carl F. Schaefer, Kenneth H. Buetow, Haleem J. Issaq, Timothy D. Veenstra

Research output: Contribution to journalArticlepeer-review

69 Scopus citations

Abstract

In this study, we utilized a multidimensional peptide separation strategy combined with tandem mass spectrometry (MS/MS) for the identification of proteins in human serum. After enzymatically digesting serum with trypsin, the peptides were fractionated using liquid-phase isoelectric focusing (IEF) in a novel ampholyte-free format. Twenty IEF fractions were collected and analyzed by reversed-phase microcapillary liquid chromatography (LC)-MS/MS. Bioinformatic analysis of the raw MS/MS spectra resulted in the identification of 844 unique peptides, corresponding to 437 proteins. This study demonstrates the efficacy of ampholyte-free peptide autofocusing, which alleviates peptide losses in ampholyte removal strategies. The results show that the separation strategy is effective for high-throughput characterization of proteins from complex proteomic mixtures.

Original languageEnglish (US)
Pages (from-to)128-133
Number of pages6
JournalElectrophoresis
Volume25
Issue number1
DOIs
StatePublished - Jan 1 2004
Externally publishedYes

Keywords

  • Ampholyte-free isoelectric focusing
  • Microcapillary liquid chromatography
  • Proteome
  • Serum
  • Tandem mass spectrometry

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

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