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

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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 (sLex) oligosaccharide and a 3′-sialyl N-acetyllactosamine (3′-SLN) on a serine (S) residue. In the negative-ion mode, the CID MS2 spectra of O-glycopeptides showed a relatively abundant glycoside-bond cleavage between the core N-acetylglucosamine (GlcNAc) and serine (S) that yields deprotonated C3-type fragment ions of O-glycan and deprotonated Z0-type peptide ions. The structure of the sLex (3′-SLN) oligosaccharide was simply assigned by comparing the CID MS3 spectrum derived from the C3-type fragment ion with the CID MS2 spectra of the sLex and sLea (3′- and 6′-SLN) standards (i.e., negative-ion MSn spectral matching). The amino acid sequence of the peptide including the glycosylation site was determined from the ECD MS2 spectrum in the positive-ion mode. Copyright © 2007 John Wiley & Sons, Ltd.