Evaluating preparative isoelectric focusing of complex peptide mixtures for tandem mass spectrometry-based proteomics: A case study in profiling chromatin-enriched subcellular fractions in Saccharomyces cerevisiae

Hongwei Xie, Sricharan Bandhakavi, Timothy J Griffin

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Abstract

We have evaluated the use of free-flow electrophoresis, an emerging separation method for preparative isoelectric focusing of complex peptide mixtures, as a tool for high-throughput tandem mass spectrometry-based proteomic analysis. In this study, we investigated the ability of free-flow electrophoresis to resolve and fractionate complex peptide mixtures and also the effectiveness of using peptide isoelectric point in conjunction with peptide match probability scoring in sequence database searching. As a model system for this study, we analyzed a chromatin-enriched fraction from the yeast Saccharomyces cerevisiae. This mixture was fractionated using preparative isoelectric focusing by free-flow electrophoresis, followed by online capillary liquid chromatography electrospray tandem mass spectrometry and sequence database searching. Our results demonstrate that (1) FFE effectively resolves and fractionates complex peptide mixtures on the basis of peptide isoelectric point and (2) the introduction of peptide pI is effective in minimizing both false positive and false negative sequence matches in sequence database searching of tandem mass spectrometry data.

Original languageEnglish (US)
Pages (from-to)3198-3207
Number of pages10
JournalAnalytical chemistry
Volume77
Issue number10
DOIs
StatePublished - May 15 2005

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Yeast
Chromatin
Mass spectrometry
Peptides
Electrophoresis
Liquid chromatography
Proteomics
Isoelectric Focusing
Throughput

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title = "Evaluating preparative isoelectric focusing of complex peptide mixtures for tandem mass spectrometry-based proteomics: A case study in profiling chromatin-enriched subcellular fractions in Saccharomyces cerevisiae",
abstract = "We have evaluated the use of free-flow electrophoresis, an emerging separation method for preparative isoelectric focusing of complex peptide mixtures, as a tool for high-throughput tandem mass spectrometry-based proteomic analysis. In this study, we investigated the ability of free-flow electrophoresis to resolve and fractionate complex peptide mixtures and also the effectiveness of using peptide isoelectric point in conjunction with peptide match probability scoring in sequence database searching. As a model system for this study, we analyzed a chromatin-enriched fraction from the yeast Saccharomyces cerevisiae. This mixture was fractionated using preparative isoelectric focusing by free-flow electrophoresis, followed by online capillary liquid chromatography electrospray tandem mass spectrometry and sequence database searching. Our results demonstrate that (1) FFE effectively resolves and fractionates complex peptide mixtures on the basis of peptide isoelectric point and (2) the introduction of peptide pI is effective in minimizing both false positive and false negative sequence matches in sequence database searching of tandem mass spectrometry data.",
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AU - Bandhakavi, Sricharan

AU - Griffin, Timothy J

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