Analysis of N- and O-linked glycans from glycoproteins using MALDI-TOF mass spectrometry.
Glycosylation represents the most common of all known protein post-translational modifications. Carbohydrates can modulate the biological functions of a glycoprotein, protect a protein against hydrolysis via protease activity, and reduce or prevent aggregation of a protein. The determination of the carbohydrate structure and function in glycoproteins remains one of the most challenging tasks given to biochemists, as these molecules can exhibit complex branched structures that can differ in linkage and in the level of branching. In this review, we will present the approach followed in our laboratory for the elucidation of N- and O-glycan chains of glycoproteins. First, reduced/carboxamidomethylated glycoproteins are digested with a protease or a chemical reagent. N-Glycans are then released from the resulting peptides/glycopeptides via digestion with peptide N-glycosidase F (PNGase F). Oligosaccharides released by PNGase F are separated from peptides and glycopeptides using a C18 Sep-Pak, and their methylated derivatives are characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). O-Glycans are released by reductive elimination, which are permethylated, purified on a Sep-Pak C18 cartridge, and analyzed with MALDI-TOF-MS. Finally, to confirm the structures N-glycans released by PNGase F are characterized using MALDI-TOF-MS following on-plate sequential exoglycosidase digestions. The clean-up procedures of native and permethylated oligosaccharides for an efficient MALDI-TOF-MS analysis will also be described. This strategy was applied to calf fetuin and glycoproteins present in human serum.
Morelle
Willy
W
Unité Mixte de Recherche CNRS/USTL 8576, << Glycobiologie Structurale et Fonctionnelle >>, IFR 118, Bâtiment C9, Université des Sciences et Technologies de Lille 1, 59655, Villeneuve d'Ascq Cedex, France. willy.morelle@univ-lille1.fr
Faid
Valegh
V
Chirat
Frédéric
F
Michalski
Jean-Claude
JC
D000818:Animals; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D006023:Glycoproteins; D011134:Polysaccharides; D019163:Reducing Agents; D006026:Glycoside Hydrolases