GPI-anchored membrane proteins (GPI-APs) are a functionally and structurally diverse protein family
present in eukaryote cells. Their common feature is the anchoring mechanism via glycosylphosphatidylinositol
(GPI) to a lipid located in the extracellular leaflet of plasma membrane. Following a modification specific
proteomic strategy, GPI-APs can be selectively isolated by a combined treatment of membrane fractions with
Triton X-114 detergent and phosphatidylinositol-specific phospholipase C and/or D. The released proteins can be
further identified by liquid chromatography on-line coupled to a tandem mass spectrometer. Although the method
is proven to be highly specific, some non-GPI-proteins are also known to be released. Bioinformatics has been
regularly used to discriminate the GPI-APs among all the identified proteins with a high degree of accuracy.
Structural characterization of the GPI-anchors has been historically a laborious analytical task. Recently, HILIC
(Hydrophilic Interaction Liquid Chromatography) and titanium dioxide enrichment of GPI-modified peptides in
combination with mass spectrometry have been used to isolate and analyse GPI-anchors as well as for the
determination of the anchor attachment site. The amino acid where the previously synthesized GPI-anchor is
attached is named the omega site (ω-site). Moreover, GPI-specific diagnostic ions detected in tandem mass
spectra can potentially be used in large-scale proteomic experiments to track GPI-specific peptides in complex
mixtures. All the information obtained by the mentioned strategies has been used for the development of an
integrated computational and experimental proteomic approach designed for identification of GPI-anchored
peptides in MS/MS spectra as well as for ω-site determination.