Reducing Interferences in Glycosylation Site Mapping

Abstract

A current method to locate sites of N-linked glycosylation on a protein involves the identification of deamidated sites after releasing the glycans with peptide-Nglycosidase F (PNGase F). PNGase F deglycosylation converts glycosylated Asn residues into Asp. The 1-Da mass tag created by this process is observable by liquid chromatography-tandem mass spectrometry analysis. A potential interference to this method of N-glycosylation site mapping is the chemical deamidation of Asn residues, which occurs spontaneously and can result in false positives. Deamidation is a pHdependent process that results in the formation of iso-Asp (i-Asp) and native Asp (n-Asp) by a succinimide intermediate, whereas PNGase F deglycosylation results in the conversion of the glycosylation Asn residue into n-Asp. N-linked glycosylation sites can thus be identified by the presence of a single chromatographic peak corresponding to an n-Asp residue within the consensus sequence Asn-X-Ser/Thr, whereas sites of deamidation led to 2 chromatographic peaks resulting from the presence of n-Asp and i-Asp.

Chitinous material bioconversion by three new chitinases from the yeast Mestchnikowia pulcherrima

“…In fact, six potential N-glycosylation sites were predicted in the MpChit35 sequence, eight in MpChit38 and seven in MpChit41 (Additional File 1: Figures S1 and S2). The three proteins were treated with PNGase F, an amidase that remove N-linked oligosaccharides from glycoproteins by cleaving between the innermost GlcNAc and Asn residues [39], with different results. Thus, while MpChit35 and MpChit38 were almost completely deglycosylated since protein bands of the expected theorical weight were visualized, treatment with PNGase F poorly altered the electrophoretic profile of MpChit41 that apparently only reduced the size of the 100 kDa band to about 85-90 kDa (Fig.…”