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Characterization of N‐glycans of recombinant human thyrotropin using mass spectrometry
Author(s) -
Morelle Willy,
Donadio Sandrine,
Ronin Catherine,
Michalski JeanClaude
Publication year - 2005
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.2289
Subject(s) - exoglycosidase , chemistry , glycan , pngase f , fucosylation , glycosylation , chromatography , oligosaccharide , glycopeptide , biochemistry , tandem mass spectrometry , peptide , mass spectrometry , glycoprotein , antibiotics
Thyroid‐stimulating hormone is a vital component of the regulatory mechanism that maintains the structure and function of the thyroid gland and governs thyroid hormone release. In this paper we report the first detailed structural characterization of the N‐linked oligosaccharides of recombinant human thyroid‐stimulating hormone (rhTSH). Using a strategy combining mass spectrometric analysis and sequential exoglycosidase digestion, we have defined the structures of the N‐glycans released from recombinant human thyrotropin by peptide N‐glycosidase F. All glycans are complex‐type glycans and are mainly of the bi‐ and triantennary type with variable degrees of fucosylation and sialylation. The major non‐reducing epitope in the complex‐type glycans is: NeuAc α 2‐3Gal β 1‐4GlcNAc (sialylated LacNAc). The carbohydrate microheterogeneity at the three glycosylation sites was studied using reversed‐phase high‐performance liquid chromatography (RP‐HPLC), concanavalin A affinity chromatography and mass spectrometric techniques, including both matrix‐assisted laser desorption/ionization (MALDI) and electrospray. rhTSH was reduced, carboxymethylated and then digested with trypsin. The mixture of peptides and glycopeptides was subjected to RP‐HPLC and the structures of the glycopeptides were determined by MALDI in conjunction with on‐target exoglycosidase digestions. After PNGase F digestion, the peptide moiety of the glycopeptide was determined by the presence of the b‐ and y‐series ions derived from its amino acid sequence in the quadrupole time‐of‐flight tandem mass (QTOF‐MS/MS) spectrum. Glycosylation sites Asn‐ α 52 and Asn‐ α 78 contain mainly bi‐ and triantennary complex‐type glycans. Only glycosylation site Asn‐ α 52 bears fucosylated N‐glycans. Minor tetraantennary complex structures were also observed on both glycosylation sites. Profiling of the carbohydrate moieties of Asn‐ β 23 indicates a large heterogeneity. Bi‐, tri‐, and tetraantennary N‐glycans were present at this site. These data demonstrate site‐specificity of glycosylation in the α subunit but not in the β subunit of rhTSH with Asn‐ α 52 bearing essentially di‐ and triantennary glycans with or without core fucosylation and bi‐ and triantennary glycans with no core fucosylation being attached to Asn‐ α 78. Copyright © 2005 John Wiley & Sons, Ltd.