title: Direct approach for qualitative and quantitative characterization of glycoproteins using tandem mass tags and an LTQ Orbitrap XL electron transfer dissociation hybrid mass spectrometer.
abstract: The application of multiplexed isobaric tandem mass tag (TMT) labeling and an LTQ Orbitrap XL ETD (electron transfer dissociation) hybrid mass spectrometer as a direct approach for qualitative and quantitative characterization of glycoproteins is reported. Bovine fetuin was used as a model glycoprotein in this study. For online liquid chromatography-mass spectrometry (LC-MS) analysis, high-resolution, mass accurate full scan MS spectra were acquired in the Orbitrap mass analyzer followed by data-dependent tandem mass spectrometry (MS/MS) with alternating collision-induced dissociation (CID), ETD, and higher-energy collisional dissociation (HCD) scans. An additional in-source dissociation scan was used as a highly sensitive and selective detection method for eluting glycosylated peptides. By alternatively using three different dissociation methods, 23 glycoforms from all 5 corresponding glycopeptides were identified from a trypsin digest of bovine fetuin. With ETD, labile glycans were retained without any signs of carbohydrate cleavage with concurrent fragmentation of the peptide backbone. Glycosylation sites were clearly localized from the ETD fragmentation data. Glycan structure elucidation was accomplished using CID. The CID experiments generated fragment ions predominantly from cleavage of glycosidic bonds without breaking the peptide bond. Novel to this method, the TMT labeling protocol was modified and adapted for higher labeling efficiency, and a TriVersa NanoMate was used to reinfuse samples to improve ETD and HCD spectra of glycopeptides. Quantification with TMT was verified based on the HCD spectra from multiple nonglycopeptides and glycopeptides. This method can be used as a qualitative and quantitative technique for direct characterization of glycoproteins and has applicability for detection of counterfeit glycoprotein drug products.
authors.affiliation: U.S. Food and Drug Administration, CDER, DPA, St. Louis, Missouri 63101, United States. hongping.ye@fda.hhs.gov
authors.forename: Hongping
authors.initials: H
authors.lastname: Ye
authors.forename: Michael T
authors.initials: MT
authors.lastname: Boyne
authors.forename: Lucinda F
authors.initials: LF
authors.lastname: Buhse
authors.forename: John
authors.initials: J
authors.lastname: Hill
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D004579:Electron Transport / Q000502:physiology; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D008969:Molecular Sequence Data; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006023:Glycoproteins