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Analysis of glycosyl phosphatidylinositol‐anchored proteins by two‐dimensional gel electrophoresis
Author(s) -
Fivaz Marc,
Vilbois Francis,
Pasquali Christian,
van der Goot F. Gisou
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(20001001)21:16<3351::aid-elps3351>3.0.co;2-k
Subject(s) - isoelectric focusing , chemistry , gel electrophoresis , membrane protein , glycosylation , proteome , biochemistry , polyacrylamide gel electrophoresis , two dimensional gel electrophoresis , chromatography , proteomics , membrane , enzyme , gene
The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)‐anchored proteins y two‐dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent‐resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI‐anchored proteins. Although the GPI‐anchored proteins were readily separated by one‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), these proteins were undetectable on two‐dimensional (2‐D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent‐resistant membranes such as caveolin‐1 and flotillin‐1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI‐anchored proteins required cleavage of the lipid tail with phosphatidylinositol‐specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI‐anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI‐anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N ‐glycosylation. Pretreatment with N ‐glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N ‐linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray‐tandem mass spectrometry as illustrated for the GPI‐anchored protein, Thy‐1.