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Molecular & Cellular Proteomics 5:1382-1395, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

A Quantitative Analysis of Arabidopsis Plasma Membrane Using Trypsin-catalyzed ¹⁸O Labeling ^{* ,S}

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

From the Biotechnology Center and Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

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.

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