Comparative analysis of techniques to purify plasma membrane proteins.
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.
Weekes
Michael P
MP
Cambridge Institute for Medical Research, University of Cambridge, Cambridge, CB2 0XY, UK. mpw1001@cam.ac.uk
Antrobus
Robin
R
Lill
Jennie R
JR
Duncan
Lidia M
LM
Hör
Simon
S
Lehner
Paul J
PJ
D001710:Biotin / Q000378:metabolism; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics; D019809:Streptavidin / Q000378:metabolism
D008565:Membrane Proteins; D001710:Biotin; D019809:Streptavidin
J Proteome Res. 2008 Jul;7(7):3028-34
18537282
Chem Rev. 2007 Aug;107(8):3687-714
17683161
Electrophoresis. 1999 Dec;20(18):3551-67
10612281
J Proteome Res. 2006 Mar;5(3):634-42
16512679
Biochem J. 2008 Feb 1;409(3):731-40
17944601
Genomics Proteomics Bioinformatics. 2004 Feb;2(1):1-5
15629037
J Bacteriol. 2007 Nov;189(21):7819-28
17766422
Nat Protoc. 2007;2(8):1896-906
17703201
Infect Immun. 2007 Aug;75(8):3833-41
17517859
Biochem J. 2009 Apr 1;419(1):211-9, 2 p following 219
19053947
Nat Methods. 2009 May;6(5):359-62
19377485
Trends Biochem Sci. 2003 Mar;28(3):137-44
12633993
Nat Methods. 2009 Mar;6(3):207-9
19234450
Proteomics. 2004 Jul;4(7):1985-8
15221759
Anal Chem. 2004 Apr 1;76(7):1817-23
15053638
Angew Chem Int Ed Engl. 2008;47(39):7523-6
18712739
J Cell Sci. 2002 May 15;115(Pt 10):2003-9
11973342
J Proteome Res. 2009 Dec;8(12):5674-8
19848406
Biochem Biophys Res Commun. 2007 Jun 8;357(3):620-6
17449014
Mol Cell Proteomics. 2005 Dec;4(12):1968-76
16176923
Nat Biotechnol. 2009 Apr;27(4):378-86
19349973
Mol Cell Proteomics. 2009 Aug;8(8):1959-71
19457934
Nat Protoc. 2009;4(5):698-705
19373234
Nat Biotechnol. 2004 Aug;22(8):985-92
15258593