A quantitative analysis of arabidopsis plasma membrane using trypsin-catalyzed 18O labeling

Clark J. Nelson, Adrian D. Hegeman, Amy C. Harms, Michael R. Sussman

Research output: Contribution to journalArticlepeer-review

86 Scopus citations

Abstract

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 H218O or H216O, 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.

Original languageEnglish (US)
Pages (from-to)1382-1395
Number of pages14
JournalMolecular and Cellular Proteomics
Volume5
Issue number8
DOIs
StatePublished - Aug 2006

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