title: Structural analysis of a glycoprotein by liquid chromatography-mass spectrometry and liquid chromatography with tandem mass spectrometry. Application to recombinant human thrombomodulin.
abstract: Using recombinant human thrombomodulin (rhTM) expressed in Chinese hamster ovary (CHO) cells, we studied the structural analysis of a glycoprotein by liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS-MS). First, we analyzed the structure of both the O- and N-linked glycans in rhTM by oligosaccharide mapping using LC-MS equipped with a graphitized carbon column (GCC-LC-MS). Major O- and N-linked glycans were determined to be core 1 structure and fucosyl biantennary containing NeuAc(0-2) respectively. Next, the post-translational modifications and their heterogeneities, including the site-specific glycosylation, were analyzed by mass spectrometric peptide/glycopeptide mapping of trypsin-digested rhTM and precursor-ion scanning. Precursor-ion scanning was successful in the detection of five glycopeptides. Four N-glycosylation sites and their site-specific carbohydrate heterogeneity were determined by their mass spectra. O-Glycosylation could be estimated on the basis of its mass spectrum. We were able to identify partial beta-hydroxylation on Asn324 and Asn439, and O-linked glucose on Ser287 from the peptide/glycopeptide map and their mass spectra. We demonstrated that a sequential analysis of LC-MS and LC-MS-MS are very useful for the structural analysis of O- and N-linked glycans, polypeptides, and post-translational modifications and their heterogeneities, including site-specific glycosylation in a glycoprotein. Our method can be applied to a glycoprotein in biological samples.
authors.affiliation: Division of Biological Chemistry and Biologicals, National Institute of Health Science, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Satsuki
authors.initials: S
authors.lastname: Itoh
authors.forename: Nana
authors.initials: N
authors.lastname: Kawasaki
authors.forename: Miyako
authors.initials: M
authors.lastname: Ohta
authors.forename: Takao
authors.initials: T
authors.lastname: Hayakawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D006900:Hydroxylation; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018180:Thrombomodulin / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D018180:Thrombomodulin; D014357:Trypsin