Systematic Evaluation of Affinity Enrichment Methods for O‑GlcNAc Proteomics

O-Linked β-N-acetylglucosamine
(O-GlcNAc)
modification (i.e., O-GlcNAcylation) on proteins plays critical roles
in the regulation of diverse biological processes. However, protein
O-GlcNAcylation analysis, especially at a large scale, has been a
challenge. So far, a number of enrichment materials and methods have
been developed for site-specific O-GlcNAc proteomics in different
biological settings. Despite the presence of multiple methods, their
performance for the O-GlcNAc proteomics is largely unclear. In this
work, by using the lysates of PANC-1 cells (a pancreatic cancer cell
line), we provided a head-to-head comparison of three affinity enrichment
methods and materials (i.e., antibody, lectin AANL6, and an OGA mutant)
for site-specific O-GlcNAc proteomics. The enriched peptides were
analyzed by HCD product-dependent EThcD (i.e., HCD-pd-EThcD) mass
spectrometry. The resulting data files were processed by three different
data analysis packages (i.e., Sequest HT, Byonic, and FragPipe). Our
data suggest that each method captures a subpopulation of the O-GlcNAc
proteins. Besides the enrichment methods, we also observe complementarity
between the different data analysis tools. Thus, combining different
approaches holds promise for enhanced coverage of O-GlcNAc proteomics.