Serially coupled microcolumn reversed phase liquid chromatography for shotgun proteomic analysis

Dingyin Tao, Guijie Zhu, Liangliang Sun, Junfeng Ma, Zhen Liang, Weibing Zhang, Lihua Zhang, Yukui Zhang

Research output: Contribution to journalArticle

Abstract

Microcolumn RPLC (μRPLC) is one of the optimum separation modes for shotgun proteomic analysis. To identify as many proteins as possible by MS/MS, the improvement on separation efficiency and peak capacity of μRPLC is indispensable. Although the increase in column length is one of the effective solutions, the preparation of a long microcolumn is rather difficult due to the high backpressure generated during the packing procedure. In our recent work, through connecting microcolumns of 5, 10, and 15 cm length via unions with minimal dead volume, long microcolumns with length up to 30 cm were obtained, with which 318 proteins were identified from proteins extracted from Escherichia coli by μRPLC-ESI MS/MS, and similar distributions of Mw and pI were found with single and various coupled microcolumns. Furthermore, by using MS/MS with improved sensitivity, with such a serially coupled 30 cm long microcolumn, 1692 proteins were identified within 7 h from rat brain tissue, with false positive rate (FPR) <1%. All these results demonstrated that serially couple microcolumns might be of great promising to improve the separation capacity of μRPLC in shotgun proteomic analysis.

Original languageEnglish (US)
Pages (from-to)2029-2036
Number of pages8
JournalProteomics
Volume9
Issue number7
DOIs
StatePublished - Apr 1 2009

Keywords

  • Serially coupled microcolumn
  • Shotgun proteome analysis
  • μRPLC MS/MS

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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