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Scientific article
English

Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis

Published inElectrophoresis, vol. 21, no. 16, p. 3351-3356
Publication date2000-10
Abstract

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.

eng
Keywords
  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Cricetinae
  • Electrophoresis, Gel, Two-Dimensional / methods
  • Glycosylphosphatidylinositols / chemistry
  • Mass Spectrometry / methods
  • Molecular Sequence Data
Citation (ISO format)
FIVAZ, Marc et al. Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis. In: Electrophoresis, 2000, vol. 21, n° 16, p. 3351–3356. doi: 10.1002/1522-2683(20001001)21:16<3351::AID-ELPS3351>3.0.CO;2-K
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ISSN of the journal0173-0835
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