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J Biomol Tech. 2010 September; 21(3 Suppl): S54–S55.
PMCID: PMC2918005

Comparative Analysis of Techniques to Purify Plasma Membrane Proteins

R. Antrobus,1 J. Lill,2 L. Duncan,1 S. Hoer,1 and P.J. Lehner1
1Cambridge Institute for Medical Research, Wellcome Trust / MRC building, Hills Road, Cambridge, United Kingdom;
2Genentech, Inc., South San Francisco, CA, United States

Abstract

RP-107

We compared existing proteomic methods for identification of the maximum number of plasma membrane proteins. Using tryptic digestion and single 4h MS runs on an LTQ Orbitrap, we compared the following techniques to determine the total number (round brackets) and percentage purity [square brackets] of plasma membrane (PM) proteins identified: (a) 4% SDS whole cell lysate (84-112) [9-13%], (b) crude membrane preparation (111 - 104) [17-20%], (c) Biotinylation and Streptavidin pulldown with NHS-SS-Biotin (78-115) [27-31%], (d) Biotinylation and Streptavidin pulldown with Biocytin Hydrazide (41-54) [59-85%], (e) Biotinylation and Streptavidin pulldown with Aminooxy-Biotin (reference 1) (120) [65%]. Data processing was performed with MaxQuant. Gene Ontology (GO) terms were examined to determine plasma membrane localisation. We increased the overall number of proteins identified by employing the recently described StageTip-based anion exchange (SAX) fractionation (2), which led to a 2-3 fold increase in protein identifications. Combining technique (e) with SAX increased the number of proteins identified to 281 [54%]. Analysis of GO terms describing these proteins identifies a large subset of proteins integral to the membrane with no subcellular assignment. These are likely to be of plasma membrane location and bring the total PM protein identifications to 364 [69%]. Selective biotinylation of the cell surface using Aminooxy-biotin in combination with SAX fractionation is a useful method for identification of glycosylated plasma membrane proteins. (1) Zeng Y, Nat. Methods 2009, (2) Wisniewski JR, J. Prot. Research 2009.


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