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

doi:10.1002/elps.200305700

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^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-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 language	English
Pages (from-to)	128-133
Number of pages	6
Journal	Electrophoresis
Volume	25
Issue number	1
DOIs	https://doi.org/10.1002/elps.200305700
State	Published - Jan 2004
Externally published	Yes

Keywords

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

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10.1002/elps.200305700

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

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.",

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AU - Xiao, Zhen

AU - Conrads, Thomas P.

AU - Lucas, David A.

AU - Janini, George M.

AU - Schaefer, Carl F.

AU - Buetow, Kenneth H.

AU - Issaq, Haleem J.

AU - Veenstra, Timothy D.

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AB - 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.

KW - Ampholyte-free isoelectric focusing

KW - Microcapillary liquid chromatography

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KW - Tandem mass spectrometry

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

Abstract

Keywords

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