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  4. Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis
 
research article

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

Fivaz, M.
•
Vilbois, F.
•
Pasquali, C.
Show more
2000
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.

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Type
research article
DOI
10.1002/1522-2683(20001001)21:16<3351::AID-ELPS3351>3.0.CO;2-K
Author(s)
Fivaz, M.
Vilbois, F.
Pasquali, C.
van der Goot, F. G.  
Date Issued

2000

Published in
Electrophoresis
Volume

21

Issue

16

Start page

3351

End page

6

Subjects

Amino Acid Sequence

•

Animals

•

Cell Line

•

Cricetinae

•

Electrophoresis

•

Gel

•

Two-Dimensional/*methods

•

Glycosylphosphatidylinositols/*chemistry

•

Mass Spectrometry/methods

•

Molecular Sequence Data

Note

Department of Biochemistry, University of Geneva, Switzerland.

Editorial or Peer reviewed

REVIEWED

Written at

OTHER

EPFL units
VDG  
Available on Infoscience
January 30, 2009
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/34621
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