Shotgun proteomic analysis of the microsomal fraction of eukaryotic cells using a two-dimensional reversed-phase x ion-pair reversed-phase HPLC setup

Martin Wörner, Katja Melchior, Nathanaël Delmotte, Kyung Hoon Hwang, Katalin Monostory, Christian G. Huber, Rita Bernhardt

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5 Citations (Scopus)

Abstract

A RP x IP-RP HPLC separation scheme was combined with on-line ESI-IT tandem MS or off-line MALDI tandem TOF MS and applied to the analysis of eukaryotic subcellular proteomes. Previous proteomic studies [1] were complemented by the approval of the approach to eukaryotic proteomes using the fission yeast Schizosaccharomyces pombe. The major focus was set to the analysis of primary human hepatocyte microsomes, representing a compartment of high interest due to its involvement in xenobiotic detoxification and cholesterol homeostasis. Of the 588 proteins identified from two donors, 24% are involved in cholesterol homeostasis or xenobiotic/lipid metabolism. Up to 50% of the identified proteins belong to the group of membrane proteins, difficult to investigate using gel-based proteomic approaches. We further demonstrated the reproducibility and comparability of the approach and reduced the amount of sample load by almost 70% with only minor loss of information about the proteins identified in the samples. The presented study clearly demonstrates the good applicability of the experimental setup to the analysis of subcellular proteomes including large membrane fractions, where only low amounts of sample material are available.

Original languageEnglish
Pages (from-to)1165-1174
Number of pages10
JournalJournal of separation science
Volume32
Issue number8
DOIs
Publication statusPublished - Jul 13 2009

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Keywords

  • Microsomal proteins
  • Primary human hepatocytes
  • Shotgun proteomics
  • Subfractionation

ASJC Scopus subject areas

  • Analytical Chemistry
  • Filtration and Separation

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