Micro- and macro- heterogeneity of N-glycoproteome remodeling in endoplasmic reticulum stress

N-glycosylation plays essential roles in the folding, trafficking, and maturation of proteins in the secretory pathways, but how individual protein and residue glycosylation rewires under endoplasmic reticulum (ER) stress is unknown. Particularly, intact glycopeptide data that retain the connectivity between glycosylation sites and the attached glycans are needed to reveal the micro- and macro- heterogeneity of N-glycosylation sites and their permutations in stressed cells. Here, we developed an optimized magnetic polyethyleneimine boronic acid-containing scaffold (mPBA) enrichment workflow to achieve sensitive and broad enrichment of intact glycoproteins for mass spectrometry analysis, quantifying 13759 unique protein-, site-, and glycoform combinations, termed glycopeptidoforms, in normal and stressed cells while requiring only 0.1 to 0.5 mg total peptide input. The data reveals a systems-level shift in the fate of hundreds of glycoproteins. N-glycosylation changes are highly dynamic, with magnitude far exceeding protein expression changes, and showing complex protein-, site-, and glycan-specific granularity. Individual glycoform reconfigurations can be observed that suggest lesions within specific steps in protein maturation and trafficking pathways. Mannose trimming is disrupted across multiple proteins and cell states, suggesting a central feature of ER stress mediated glycoproteome remodeling. Together, these results reveal molecular details into the remodeling of protein secretory pathways upon ER stress and highlight the utility of mPBA for sensitive N-glycoproteomics studies.