Pharmaceutical Sciences Faculty Publications

Direct Ampholyte-Free Liquid-Phase Isoelectric Peptide Focusing: Application to the Human Serum Proteome

Document Type

Article

Publication Date

1-1-2004

Journal Title

Electrophoresis

ISSN

0173-0835

Volume

25

Issue

1

First Page

128

Last Page

133

DOI

10.1002/elps.200305700

PubMed ID

14730577

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 (microLC)-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.

Keywords

Blood proteins, humans, isoelectric focusing, mass spectrometry, peptide fragments, proteome, proteomics

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