Quantitative Analysis of Cell Surface Membrane Proteins Using Membrane-Impermeable Chemical Probe Coupled with 18O Labeling

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• 作者：Haizhen Zhang ; Roslyn N. Brown ; Wei-Jun Qian ; Matthew E. Monroe ; Samuel O. Purvine ; Ronald J. Moore ; Marina A. Gritsenko ; Liang Shi ; Margaret F. Romine ; James K. Fredrickson ; Ljiljana Paa-Toli ; Richar
• 刊名：Journal of Proteome Research
• 出版年：2010
• 出版时间：May 7, 2010
• 年：2010
• 卷：9
• 期：5
• 页码：2160-2169
• 全文大小：328K
• 年卷期：v.9,no.5(May 7, 2010)
• ISSN：1535-3907

文摘

We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope ¹⁸O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a Gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and extracellular electron receptors. LC/MS/MS analysis resulted in the identification of about 400 proteins with 79% of them being predicted to be membrane localized. Quantitative aspects of the membrane enrichment were shown by peptide level ¹⁶O and ¹⁸O labeling of proteins from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) prior to LC-MS analysis. Using a chemical probe labeled pure protein as an internal standard for normalization, the quantitative data revealed reduced abundances in ΔgspD mutant cells of many outer membrane proteins including the outer membrane c-type cytochromes OmcA and MtrC, in agreement with a previous report that these proteins are substrates of the type II secretion system.