Stable isotope labeling of N-glycosylated peptides by enzymatic deglycosylation for mass spectrometry-based glycoproteomics.

Protein glycosylation is one of the most common and crucial post-translational modifications that regulates many biological processes. Because abnormal glycosylation is also associated with various pathologies, including cancer, and inflammatory and degenerative diseases, technology for comprehensive analysis of glycoproteins, or glycoproteomics, is important not only for biological studies but also for biomedical and clinical research, including the discovery of biomarkers for disease diagnosis, prognosis, and therapeutic response to drugs. Here, we describe a protocol for peptide-N-glycanase-mediated (18)O labeling of N-glycosylated peptides, termed "isotope-coded glycosylation site-specific tagging." Coupled with advanced mass spectrometry-based proteomics technology, this method facilitates the identification of hundreds to thousands of N-glycoproteins, coupled with their sites of glycosylation, from a complex biological mixture.