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Submitted on December 14, 2005
Accepted on April 24, 2006

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

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

Biotechnology Center, University of Wisconsin, Madison, WI 53706

Corresponding Author: msussman{at}wisc.edu

Typical mass spectrometric-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 QTOF 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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