Quantitative Analysis of Cell Surface Membrane Proteins Using Membrane-Impermeable Chemical Probe Coupled with 18O Labeling
datasetposted 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
Datasets usually provide raw data for analysis. This raw data often comes in spreadsheet form, but can be any collection of data, on which analysis can be performed.
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.