Enzymatic/chemical release of O-glycans allowing MS analysis at high sensitivity.
As the role of O-linked oligosaccharides have been demonstrated to be increasingly important in numerous medical conditions, it is imperative to develop new techniques allowing their analysis at high sensitivity. While mass spectrometry (MS) provides adequate measurements of important O-linked oligosaccharides glycans and their profiles, the release from glycoproteins has not been sufficiently addressed for the needs of biomedical applications. This work describes a new strategy, involving the combination of a complete enzymatic degradation with a chemical release during the solid-phase permethylation of O-linked oligosaccharides. The analytical data implicate highly effective cleavage from the serine and threonine (but not arginine) residues, during permethylation. Tandem MS analyses confirmed these observations for model glycoproteins. Comparative measurements through isotopic labeling MS show this approach to be vastly superior over the previously used cleavage procedures.
Goetz
John A
JA
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
Novotny
Milos V
MV
Mechref
Yehia
Y
D002240:Carbohydrate Sequence; D004798:Enzymes / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008049:Lipase / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010100:Oxygen; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D011402:Pronase / Q000378:metabolism; D015203:Reproducibility of Results
D004798:Enzymes; D006023:Glycoproteins; D011134:Polysaccharides; D008049:Lipase; D011402:Pronase; D010100:Oxygen