A quantitative analysis of Arabidopsis plasma membrane using trypsin-catalyzed (18)O labeling

Typical mass spectrometry-based protein lists from purified fractions are confounded by the absence of tools for evaluating contaminants. In this report, we compare the results of a standard survey experiment using an ion trap mass spectrometer with those obtained using dual isotope labeling and a Q-TOF mass spectrometer to quantify the degree of enrichment of proteins in purified subcellular fractions of Arabidopsis plasma membrane. Incorporation of a stable isotope, either H(2)(18)O or H(2)(16)O, during trypsinization allowed relative quantification of the degree of enrichment of proteins within membranes after phase partitioning with polyethylene glycol/dextran mixtures. The ratios allowed the quantification of 174 membrane-associated proteins with 70 showing plasma membrane enrichment equal to or greater than ATP-dependent proton pumps, canonical plasma membrane proteins. Enriched proteins included several hallmark plasma membrane proteins, such as H(+)-ATPases, aquaporins, receptor-like kinases, and various transporters, as well as a number of proteins with unknown functions. Most importantly, a comparison of the datasets from a sequencing "survey" analysis using the ion trap mass spectrometer with that from the quantitative dual isotope labeling ratio method indicates that as many as one-fourth of the putative survey identifications are biological contaminants rather than bona fide plasma membrane proteins.