title: Structural analysis of O-glycopeptides employing negative- and positive-ion multi-stage mass spectra obtained by collision-induced and electron-capture dissociations in linear ion trap time-of-flight mass spectrometry.
abstract: Structural analyses of various glycans attached to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on mass spectrometry (MS) combining both collision-induced dissociation (CID) and electron-capture dissociation (ECD) in the positive- and negative-ion modes has been proposed as a simple and direct method of assigning an O-glycan without releasing it from the peptide and of determining the amino acid sequence of the peptide and glycosylation site. The instrument used is an electrospray ionization (ESI) linear ion trap (LIT) time-of-flight (TOF) mass spectrometer with tandem LITs for CID by He gas and ECD. The proposed approach was tested with two synthetic O-glycopeptides binding a sialyl Lewis x (sLe(x)) oligosaccharide and a 3'-sialyl N-acetyllactosamine (3'-SLN) on a serine (S) residue. In the negative-ion mode, the CID MS(2) spectra of O-glycopeptides showed a relatively abundant glycoside-bond cleavage between the core N-acetylglucosamine (GlcNAc) and serine (S) that yields deprotonated C(3)-type fragment ions of O-glycan and deprotonated Z(0)-type peptide ions. The structure of the sLe(x) (3'-SLN) oligosaccharide was simply assigned by comparing the CID MS(3) spectrum derived from the C(3)-type fragment ion with the CID MS(2) spectra of the sLe(x) and sLe(a) (3'- and 6'-SLN) standards (i.e., negative-ion MS(n) spectral matching). The amino acid sequence of the peptide including the glycosylation site was determined from the ECD MS(2) spectrum in the positive-ion mode.
authors.affiliation: Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan. deguchi@glyco.sci.hokudai.ac.jp
authors.forename: Kisaburo
authors.initials: K
authors.lastname: Deguchi
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ito
authors.forename: Takashi
authors.initials: T
authors.lastname: Baba
authors.forename: Atsumu
authors.initials: A
authors.lastname: Hirabayashi
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Masataka
authors.initials: M
authors.lastname: Fumoto
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hinou
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
mesh_terms: D000606:Amino Sugars / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry*; D010449:Peptide Mapping; D000080506:Sialyl Lewis X Antigen; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D000606:Amino Sugars; D006020:Glycopeptides; D009844:Oligosaccharides; D000080506:Sialyl Lewis X Antigen; C032269:sialyl-N-acetyllactosamine