Comparative analysis of techniques to purify plasma membrane proteins - PubMed (original) (raw)

Comparative Study

Comparative analysis of techniques to purify plasma membrane proteins

Michael P Weekes et al. J Biomol Tech. 2010 Sep.

Abstract

The aim of this project was to identify the best method for the enrichment of plasma membrane (PM) proteins for proteomics experiments. Following tryptic digestion and extended liquid chromatography-tandem mass spectrometry acquisitions, data were processed using MaxQuant and Gene Ontology (GO) terms used to determine protein subcellular localization. The following techniques were examined for the total number and percentage purity of PM proteins identified: (a) whole cell lysate (total number, 84-112; percentage purity, 9-13%); (b) crude membrane preparation (104-111; 17-20%); (c) biotinylation of surface proteins with N-hydroxysulfosuccinimydyl-S,S-biotin and streptavidin pulldown (78-115; 27-31%); (d) biotinylation of surface glycoproteins with biocytin hydrazide and streptavidin pulldown (41-54; 59-85%); or (e) biotinylation of surface glycoproteins with amino-oxy-biotin (which labels the sialylated fraction of PM glycoproteins) and streptavidin pulldown (120; 65%). A two- to threefold increase in the overall number of proteins identified was achieved by using stop and go extraction tip (StageTip)-based anion exchange (SAX) fractionation. Combining technique (e) with SAX fractionation increased the number of proteins identified to 281 (54%). Analysis of GO terms describing these proteins identified a large subset of proteins integral to the membrane with no subcellular assignment. These are likely to be of PM location and bring the total PM protein identifications to 364 (68%). This study suggests that selective biotinylation of the cell surface using amino-oxy-biotin in combination with SAX fractionation is a useful method for identification of sialylated PM proteins.

Keywords: NHS-SS-biotin; biotin; glycoprotein; proteomics; sialic acid.

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Conflict of interest statement

There are no conflicts of interest.

Figures

FIGURE 1

FIGURE 1

Workflow illustrating additional methods attempted to increase purity of PM preparation using NHS-SS-biotinylation of the cell surface.

FIGURE 2

FIGURE 2

SAX fractionation of peptides generated by using the amino-oxy-biotin technique leads to a substantial increase in the number of peptides per protein identified. We compared data for the two amino-oxy-biotin preparations shown in Table 3. Prep 1—single, 4-h MS run, no fractionation; Prep 2—duplicate, 4-h MS run on six peptide fractions.

FIGURE 3

FIGURE 3

A substantial number of proteins isolated using amino-oxy-biotin are identified by GO as integral to the membrane but have no subcellular assignment. These are likely to be PM proteins. Separated GO terms are shown for a representative sample of the full list of proteins identified in amino-oxy-biotin Prep 2, Table 3. Seventy-one out of 539 total proteins had a very short GO descriptor (examples indicated by arrows), identifying the protein as integral to membrane but with no subcellular assignment. These are likely to be PM proteins.

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