Structural analysis of a glycoprotein by liquid chromatography-mass spectrometry and liquid chromatography with tandem mass spectrometry. Application to recombinant human thrombomodulin.
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.
Itoh
Satsuki
S
Division of Biological Chemistry and Biologicals, National Institute of Health Science, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Kawasaki
Nana
N
Ohta
Miyako
M
Hayakawa
Takao
T
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
D011994:Recombinant Proteins; D018180:Thrombomodulin; D014357:Trypsin