Characterization of N‐glycans of recombinant human thyrotropin using mass spectrometry

Abstract

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.

Analysis of protein glycosylation by mass spectrometry

“…Recently, we have successfully applied this approach to the analysis of different glycoproteins, including galactommanoproteins of Aspergillus fumigatus 6 , excreted/secreted glycoproteins of the parasite Giardia intestinalis 7 , bovine lysosomal a-mannosidase 5 and recombinant human thyrotropin 8 . We have also identified acquired modifications of glycosylation by applying this approach to the serum of patients with cirrhosis 9 .…”

“…m CRITICAL STEP Prepare the trypsin solution just before use. Since PNGase F enzyme does not release any N-glycans when glycosylation sites are on the first or last amino acid residue of a peptide, the protease must be carefully chosen 8 . For this reason, when PNGase F digestion is performed on a glycoprotein, the amino acid sequence of this glycoprotein must be known.…”

“…1). This robust and highly sensitive mass spectrometric approach for characterizing the glycosylation pattern of proteins, cells, tissues and physiological fluids allows a rapid characterization of the glycoforms as well as their relative quantitation [5][6][7][8][9][10] .…”

Sequential enrichment of sulfated glycans by strong anion-exchange chromatography prior to mass spectrometric measurements

“…This glycoprotein features mono-and disulfated glycan structures ( Figure 3a) [2,33,34]. N-glycans from bTSH were released, permethylated, and sequentially enriched according to the optimized procedure described above.…”

“…As the spin-columns are individually optimized for each sulfation state, this system can be used to selectively enrich structures with a given sulfation state. We applied this method to both sulfated oligosaccharide standards as well as the Nglycans isolated from bovine thyroid-stimulating hormone (bTSH), which is a well-studied glycoprotein expressed in bovine anterior pituitary [2,33,34].…”