Systematic\nEvaluation of Affinity Enrichment Methods\nfor O‑GlcNAc Proteomics

O-Linked β-<i>N</i>-acetylglucosamine\n(O-GlcNAc)\nmodification (i.e., O-GlcNAcylation) on proteins plays critical roles\nin the regulation of diverse biological processes. However, protein\nO-GlcNAcylation analysis, especially at a large scale, has been a\nchallenge. So far, a number of enrichment materials and methods have\nbeen developed for site-specific O-GlcNAc proteomics in different\nbiological settings. Despite the presence of multiple methods, their\nperformance for the O-GlcNAc proteomics is largely unclear. In this\nwork, by using the lysates of PANC-1 cells (a pancreatic cancer cell\nline), we provided a head-to-head comparison of three affinity enrichment\nmethods and materials (i.e., antibody, lectin AANL6, and an OGA mutant)\nfor site-specific O-GlcNAc proteomics. The enriched peptides were\nanalyzed by HCD product-dependent EThcD (i.e., HCD-pd-EThcD) mass\nspectrometry. The resulting data files were processed by three different\ndata analysis packages (i.e., Sequest HT, Byonic, and FragPipe). Our\ndata suggest that each method captures a subpopulation of the O-GlcNAc\nproteins. Besides the enrichment methods, we also observe complementarity\nbetween the different data analysis tools. Thus, combining different\napproaches holds promise for enhanced coverage of O-GlcNAc proteomics.