Pharmaceutical Sciences Faculty Publications

Development of a Two-Dimensional Protein-Peptide Separation Protocol for Comprehensive Proteome Measurements

Document Type

Article

Publication Date

4-5-2003

Journal Title

Journal of Chromatography B

ISSN

1570-0232

Volume

787

Issue

1

First Page

43

Last Page

51

DOI

10.1016/s1570-0232(02)00616-5

PubMed ID

12659732

Abstract

We have developed an effective two-dimensional fractionation protocol of complex proteome mixtures that extends the ability to conduct more comprehensive proteome measurements. A sample containing intact proteins extracted from Saccharomyces cerevisiae was fractionated by liquid phase isoelectric focusing, followed by tryptic digestion and solid-phase extraction (SPE) clean-up and reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS-MS) of the resultant peptides. The clean-up step is designed to desalt the fractions and rid them of urea and ampholytes prior to analysis by LC-MS-MS. Fifty milligrams of protein were separated into 20 fractions by liquid-phase isoelectric focusing, spanning a pH range of 3-10. The effectiveness of the removal of ampholytes was monitored by capillary zone electrophoresis and LC-MS-MS. The ability to analyze all of the 20 fractions without any noticeable decrease in the separation efficiency demonstrates the overall effectiveness of the SPE clean-up step. The results show that the separation strategy is effective for high throughput characterization of proteins from complex proteomic mixtures.

Keywords

Electrophoresis, polyacrylamide gel, peptides, proteins, proteome

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