Characterization of N-glycans of recombinant human thyrotropin using mass spectrometry.

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: NeuAcalpha2-3Galbeta1-4GlcNAc (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-alpha52 and Asn-alpha78 contain mainly bi- and triantennary complex-type glycans. Only glycosylation site Asn-alpha52 bears fucosylated N-glycans. Minor tetraantennary complex structures were also observed on both glycosylation sites. Profiling of the carbohydrate moieties of Asn-beta23 indicates a large heterogeneity. Bi-, tri-, and tetraantennary N-glycans were present at this site. These data demonstrate site-specificity of glycosylation in the alpha subunit but not in the beta subunit of rhTSH with Asn-alpha52 bearing essentially di- and triantennary glycans with or without core fucosylation and bi- and triantennary glycans with no core fucosylation being attached to Asn-alpha78.

Morelle W ，Donadio S ，Ronin C ，Michalski JC ... -  《RAPID COMMUNICATIONS IN MASS SPECTROMETRY》

Site-specific glycosylation analysis of the bovine lysosomal alpha-mannosidase.

Faid V ，Evjen G ，Tollersrud OK ，Michalski JC ，Morelle W ... -  《GLYCOBIOLOGY》

Analysis of the site-specific N-glycosylation of beta1,6 N-acetylglucosaminyltransferase V.

Kamar M ，Alvarez-Manilla G ，Abney T ，Azadi P ，Kumar Kolli VS ，Orlando R ，Pierce M ... -  《GLYCOBIOLOGY》

N-glycans of recombinant human acid alpha-glucosidase expressed in the milk of transgenic rabbits.

Jongen SP ，Gerwig GJ ，Leeflang BR ，Koles K ，Mannesse ML ，van Berkel PH ，Pieper FR ，Kroos MA ，Reuser AJ ，Zhou Q ，Jin X ，Zhang K ，Edmunds T ，Kamerling JP ... -  《GLYCOBIOLOGY》

Site-specific characterization of the N-linked glycans of murine prion protein by high-performance liquid chromatography/electrospray mass spectrometry and exoglycosidase digestions.

Stimson E ，Hope J ，Chong A ，Burlingame AL ... -  《BIOCHEMISTRY》