title: Determination of a sugar chain and its linkage site on a glycoprotein TIME-EA4 from silkworm diapause eggs by means of LC-ESI-Q-TOF-MS and MS/MS.
abstract: The electrospray ionization (ESI)-tandem quadrupole/orthogonal-acceleration time-of-flight (Q-TOF) mass spectrometer combined with the nano-HPLC system was utilized to determine the glycosylation site and the glycan structure in glycoprotein TIME-EA4 (EA4) from Bombyx diapause eggs. LC-MS analysis of EA4 and deglycosylated EA4 indicated that the carbohydrate moiety of EA4 has the mass of 730.58 Da. Then, EA4 was digested with trypsin and chymotrypsin to identify the glycosylated peptide. The peptide fragment from G1y21 to Phe25 was found to carry the carbohydrate moiety. LC-MS/MS analysis of this peptide fragment revealed the sequence of the attached oligosaccharide and the glycosylation site at the same time. The present methodology utilizing the combination of the nano-HPLC system and a highly sensitive Q-TOF mass spectrometer is demonstrated to be quite effective for analyses of glycoproteins of relatively low purity and limited availability from natural sources.
authors.affiliation: Laboratorv of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, 464-8601, Nagoya, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Kurahashi
authors.forename: Asaka
authors.initials: A
authors.lastname: Miyazaki
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Murakami
authors.forename: Sathorn
authors.initials: S
authors.lastname: Suwan
authors.forename: Thomas
authors.initials: T
authors.lastname: Franz
authors.forename: Minoru
authors.initials: M
authors.lastname: Isobe
authors.forename: Naoki
authors.initials: N
authors.lastname: Tani
authors.forename: Hidenori
authors.initials: H
authors.lastname: Kai
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D012831:Bombyx / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis* / Q000737:chemistry*; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D002918:Chymotrypsin / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010063:Ovum / Q000737:chemistry*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D014357:Trypsin / Q000378:metabolism
chemical_list: D002241:Carbohydrates; D006023:Glycoproteins; D002918:Chymotrypsin; D014357:Trypsin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase