Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling.
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 (18)O 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 (16)O and (18)O 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 Delta gspD 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.
Zhang
Haizhen
H
Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Brown
Roslyn N
RN
Qian
Wei-Jun
WJ
Monroe
Matthew E
ME
Purvine
Samuel O
SO
Moore
Ronald J
RJ
Gritsenko
Marina A
MA
Shi
Liang
L
Romine
Margaret F
MF
Fredrickson
James K
JK
Pasa-Tolić
Ljiljana
L
Smith
Richard D
RD
Lipton
Mary S
MS
D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D016000:Cluster Analysis; D007553:Isotope Labeling / Q000379:methods*; D009154:Mutation; D010103:Oxygen Isotopes / Q000378:metabolism*; D018272:Porins / Q000235:genetics; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D020592:Shewanella / Q000737:chemistry / Q000235:genetics; D013388:Succinimides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods
D001425:Bacterial Outer Membrane Proteins; D010103:Oxygen Isotopes; D018272:Porins; D020543:Proteome; D013388:Succinimides; D001710:Biotin