A Quantitative Analysis of Arabidopsis Plasma Membrane Using Trypsin-catalyzed 18O Labeling

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

doi:10.1074/mcp.m500414-mcp200

Clark J Nelson, Adrian D Hegeman + Show 2 more

Open Access

https://doi.org/10.1074/mcp.m500414-mcp200

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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 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.

Highlights

Typical mass spectrometry-based protein lists from purified fractions are confounded by the absence of tools for evaluating contaminants
Sample Preparation—Arabidopsis thaliana ecotype Columbia was grown at 22 °C in 24-h light in liquid culture consisting of 0.5% (w/v) MES, 2.15% (w/v) Murashige and Skoog salts, and 1% (w/v) sucrose
Ion trap data were analyzed except that MS tolerance was set to Ϯ1.5 Da, MS/MS tolerance was set to Ϯ0.8 Da, and there was no allowance for 18Olabeled residues

Summary

EXPERIMENTAL PROCEDURES

Materials—All reagents were purchased from Sigma/Aldrich unless otherwise noted. Sample Preparation—Arabidopsis thaliana ecotype Columbia was grown at 22 °C in 24-h light in liquid culture consisting of 0.5% (w/v) MES (pH 5.7), 2.15% (w/v) Murashige and Skoog salts, and 1% (w/v) sucrose. Digests used in the ion trap plasma membrane surveys were conducted as for dual isotope-labeled quantitation except that 750 ␮g of upper phase protein were used and not combined with lower phase digests. Ion trap data were analyzed except that MS tolerance was set to Ϯ1.5 Da, MS/MS tolerance was set to Ϯ0.8 Da, and there was no allowance for 18Olabeled residues For both datasets, peptides scoring above the 1% FP threshold were considered. For observations with an R2 value of 0.8 or greater for both regressions, their normalized intensities could be used to calculate a heavy to light ratio using the equation below that is similar to a method described previously [20] In this equation, P0, P2, and P4 represent the measured intensities for isotopes of the zero, one, and two heavy oxygen incorporation events (monoisotopic, ϩ2, and ϩ4 isotopic peaks), respectively. Microarray experiments were queried at the Arabidopsis Membrane Protein Library website (www.cbs.umn.edu/arabidopsis/)

RESULTS

TABLE III Vacuolar proteins

TABLE IV ER proteins

DISCUSSION

TABLE VII Contaminants