Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis.
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.
Fivaz
M
M
Department of Biochemistry, University of Geneva, Switzerland.
Vilbois
F
F
Pasquali
C
C
van der Goot
F G
FG
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data
D017261:Glycosylphosphatidylinositols