Reverse-phase capillary high performance liquid chromatography/high performance electrospray ionization mass spectrometry: an essential tool for the characterization of complex glycoprotein digests

Abstract

The B-domain of recombinant human Factor VIII comprises 909 amino acids and is extensively N- and O-glycosylated, in that at least 20 different sites are occupied by numerous carbohydrate structures. This domain was incubated with trypsin and subjected to liquid chromatography electrospray ionization mass spectrometry analysis, using an electrospray orthogonal acceleration time-of-flight mass spectrometer as the detector for a capillary reversed phase HPLC separation of the digest. The inherent high mass resolution afforded by this instrument provides both ion charge state determination and high accuracy mass measurement that are of significant advantage in defining such highly complex mixtures.

Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII

“…These endoglycosidases have been shown to cleave oligomannose bi and tri-antennary complex oligosaccharide structures from glycoproteins under native conditions (26,27). Complex tetraantennary structures that are found in low frequency (one of several potential sugars on only two residues the B domain of FVIII) are not cleaved by endoglycosidase Fs (5,6). The reactions were carried out at pH 6.5 or in the buffer provided by the manufacturer for 1 h at 37°C, if not stated otherwise.…”

“…A molecular weight shift was observed for lane 3 and was due to deletion of oligomannose residues and complex bi-and tri-antennary sugar structures. The largest molecular weight shifts were recorded for the highest molecular weight heavy chains that carry the maximum number of oligosaccharides (2,4,6).…”

“…The native full length protein has 25 potential N-glycosylation sites, including 19 sites in the B-domain and three each in light (two in A1, one in A2) and heavy (two in A3, one in C1) chains (4). Of these, 21 sites were identified as carrying oligosaccharides with 16-17 potential glycosylation sites occupied in the B-domain (5,6). In spite of the presence of one Asn-Xxx-Ser/Thr consensus sequence for N-linked glycosylation, the A2 domain of FVIII does not carry any oligosaccharides.…”

Characterization and analysis of a novel glycoprotein from snake venom using liquid chromatography‐electrospray mass spectrometry and Edman degradation

“…Reverse‐phase high‐performance liquid chromatography‐electrospray ionization mass spectrometry (LC‐ESI‐MS) has been shown to be a rapid and sensitive means for characterizing the primary structure of proteins and their post‐translational modifications including glycosylation [1–7]. To determine the site of glycosylation and site‐specific microheterogeneity of glycoprotein, endoproteases are usually employed to obtain the glycopeptides which are separated by HPLC and identified by electrospray mass spectrometry.…”

Identification of protein-bound carbohydrates by mass spectrometry

“…Tryptic glycopeptides invariably give doublycharged ions or ions in higher charge states. The combination of ESI-MS with LC provides a powerful tool for mixture analysis of both glycopeptides and released glycans [38,39].…”