title: Evaluation of the oligosaccharide composition of commercial follicle stimulating hormone preparations.
abstract: Glycosylation is an important PTM in proteins, and it is of paramount importance for the manufacture and efficacy of therapeutic glycoproteins. The elucidation of the glycosylation patterns is greatly hampered by the structural heterogeneity, which involves the oligosaccharide moieties, and sometimes also the protein chain. Several strategies are used to map the glycosylation state of glycoproteins: they generally involve analysis of the glycopeptides obtained upon glycoprotein proteolytic digestion, and/or analysis of the glycans, enzymatic or chemically released from the glycoproteins. These approaches are efficient in determining the total glycan content, as well as the glycan structures; nevertheless, information regarding the whole protein is lost. Recent advances in MS allowed us to directly analyze human follicle stimulating hormone (hFSH). RP-HPLC/IT-TOF MS, coupled to bioinformatic algorithms, was used to determine the glycan content of intact hFSH preparations, either urinary-derived or recombinant. More traditional and complementary analytical methods (oligosaccharide profiling, IEF, and peptide mapping analyses) were employed to compare the obtained results. Our analysis shows a predominance of highly sialylated, highly branched glycans in a urinary hFSH preparation as compared to recombinant hFSH expressed in rodent cell lines. Noteworthy, high-resolution mass spectra may allow to evaluate the glycosylation profile during the manufacturing process of different preparations.
authors.affiliation: Department of Chemical Sciences, University Federico II of Naples, Naples, Italy. alombard@unina.it
authors.forename: Angela
authors.initials: A
authors.lastname: Lombardi
authors.forename: Concetta
authors.initials: C
authors.lastname: Andreozzi
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Pavone
authors.forename: Valeria
authors.initials: V
authors.lastname: Triglione
authors.forename: Luca
authors.initials: L
authors.lastname: Angiolini
authors.forename: Paolo
authors.initials: P
authors.lastname: Caccia
mesh_terms: D002853:Chromatography, Liquid; D005640:Follicle Stimulating Hormone / Q000032:analysis / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D021122:Protein Subunits
chemical_list: D006023:Glycoproteins; D009844:Oligosaccharides; D021122:Protein Subunits; D005640:Follicle Stimulating Hormone; D006026:Glycoside Hydrolases
keywords: Glycosylation; Gonadotropin; Human follicle stimulating hormone; Liquid chromatography-mass spectrometry; N-glycan structure