Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars.
Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Assignment of intact glycan structures to specific protein attachment sites is a critical step towards elucidating the function encoded in the glycome. Previously, we developed isotope-targeted glycoproteomics (IsoTaG) as a mass-independent mass spectrometry method to characterize azide-labeled intact glycopeptides from complex proteomes. Here, we extend the IsoTaG approach with the use of alkynyl sugars as metabolic labels and employ new probes in analysis of the sialylated glycoproteome from PC-3 cells. Using an Orbitrap Fusion Tribrid mass spectrometer, we identified 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycan structures across 126 N- and 576 O-glycopeptides. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido sugars and will facilitate further studies of the glycoproteome.
Department of Chemistry, Stanford University, Stanford, CA, 94305, USA.
Christina M
CM
Woo
Department of Chemistry, Stanford University, Stanford, CA, 94305, USA.
Alejandra
A
Felix
Chemical and Systems Biology, Stanford University, Stanford, CA, 94305, USA.
Lichao
L
Zhang
Chemical and Systems Biology, Stanford University, Stanford, CA, 94305, USA.
Joshua E
JE
Elias
Department of Chemistry, Stanford University, Stanford, CA, 94305, USA. bertozzi@stanford.edu.|Howard Hughes Medical Institute, Stanford University, Stanford, CA, 94305, USA. bertozzi@stanford.edu.
Carolyn R
CR
Bertozzi
D001386:Azides / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry*; D007554:Isotopes / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry*; D040901:Proteomics
D001386:Azides; D006020:Glycopeptides; D007554:Isotopes; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid
Chemical proteomics; Glycoproteomics; LC-MS/MS; Metabolic labeling; Sialic acid
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