Highly efficient proteome analysis with combination of protein pre-fractionation by preparative microscale solution isoelectric focusing and identification by μrPLC-MS/MS with serially coupled long microcolumn

Dingyin Tao, Liangliang Sun, Guijie Zhu, Yu Liang, Zhen Liang, Lihua Zhang, Yukui Zhang

Research output: Contribution to journalArticle

Abstract

To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns.

Original languageEnglish (US)
Pages (from-to)83-89
Number of pages7
JournalJournal of Separation Science
Volume34
Issue number1
DOIs
StatePublished - Jan 1 2011

Keywords

  • Protein pre-fractionation
  • Serially coupled microcolumn
  • Shotgun proteome analysis
  • μRPLC-MS/MS

ASJC Scopus subject areas

  • Analytical Chemistry
  • Filtration and Separation

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