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

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posted on 07.05.2010 by 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 Paša-Tolić, Richard D. Smith, Mary S. Lipton
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.

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