Characterization of sialylated N- and O-glycopeptides from complex proteomes on an Orbitrap Fusion Tribrid mass spectrometer by Isotope Targeted Glycoproteomics (IsoTaG). Application to azido and alkynyl sugars

Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Correlation of the intact glycoform to the protein attachment site is a critical step to assign functional roles to specific glycoproteins. Isotope targeted glycoproteomics (IsoTaG) is a mass-independent mass spectrometry method to characterize intact, metabolically labeled glycopeptides from complex proteomes. IsoTaG was applied to conditioned media from PC-3 cells labeled with alkynyl or azido sugars to reveal the sialylated glycoproteome. Analysis on an Orbitrap Fusion Tribrid mass spectrometer resulted in characterization of 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycoforms across 126 Nand 576 O-glycopeptides. IsoTaG is an effective platform for identification of intact glycopeptides labeled by alkynyl or azido-glycans and will facilitate further studies on the role of the glycoproteome in biology.