Mass Spectrometric Identification of Glycosylphosphatidylinositol-Anchored Peptides

Glycosylphosphatidylinositol
(GPI) anchoring is a post-translational
modification widely observed among eukaryotic membrane proteins. GPI
anchors are attached to proteins via the carboxy-terminus in the outer
leaflet of the cell membrane, where GPI-anchored proteins (GPI-APs)
perform important functions as coreceptors and enzymes. Precursors
of GPI-APs (Pre-GPI-APs) contain a C-terminal hydrophobic sequence
that is involved in cleavage of the signal sequence from the protein
and addition of the GPI anchor by the transamidase complex. In order
to confirm that a given protein contains a GPI anchor, it is essential
to identify the C-terminal peptide containing the GPI-anchor modification
site (ω-site). Previously, efficient identification of GPI-anchored
C-terminal peptides by mass spectrometry has been difficult, in part
because of complex structure of the GPI-anchor moiety. We developed
a method to experimentally identify GPI-APs and their ω-sites.
In this method, a part of GPI-anchor moieties are removed from GPI-anchored
peptides using phosphatidylinositol-specific phospholipase C (PI-PLC)
and aqueous hydrogen fluoride (HF), and peptide sequence is then determined
by mass spectrometry. Using this method, we successfully identified
10 GPI-APs and 12 ω-sites in the cultured ovarian adenocarcinoma
cells, demonstrating that this method is useful for identifying efficiently
GPI-APs.