Elucidation of N-glycosylation sites on human platelet proteins: a glycoproteomic approach.
Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets.
Lewandrowski
Urs
U
Protein Mass Spectrometry and Functional Proteomics Group, Rudolf Virchow Center for Experimental Biomedicine, Versbacher Strasse 9, 97078 Wuerzburg, Germany.
Moebius
Jan
J
Walter
Ulrich
U
Sickmann
Albert
A
D000595:Amino Acid Sequence; D004815:Epidermal Growth Factor; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019007:P-Selectin / Q000737:chemistry; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D040901:Proteomics; D019700:Thrombospondin 1 / Q000737:chemistry / Q000378:metabolism
D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D019700:Thrombospondin 1; D004815:Epidermal Growth Factor