A Chemoproteomic Approach for System-Wide and Site-Specific Uncovering of Functional Protein N‑Glycosylation

Technological
advances in proteomics, including sample separation,
mass spectrometry, and searching algorithms, have empowered in-depth
discovery of protein post-translational modifications from biological
samples. However, there is still a considerable delay in systematic
research on the functional significance of these modifications. Herein,
we develop a new thermal proteomic strategy, Refined-TPP, enabling
the efficient study of protein thermostability with improved sensitivity
and increased throughput by 5-fold. Its robust performance in target
identification was demonstrated by the metabolite NADPH as well as
the drug panobinostat. We further propose glyco-dependent thermal
shift profiling (GTSP), a novel chemical proteomic methodology, to
systematically examine the effects of site-specific modifications
on the thermal stability of native proteins. Finally, we probed 208
functionally important N-glycosites mapping 113 proteins and elucidated
their pivotal roles in protein functions, including protein stability,
subcellular localization, and enzyme activity. This globally biophysical
assay bridges the gap between structural modifications and their functional
impacts on proteins and provides here a robust platform for the first
time to effectively interrogate functional implications of N-glycosylation.
It is also readily applicable in a high-throughput and unbiased manner
to further investigations into diverse objects, including drug-target
screening, protein interactions, and other functional PTM exploration.