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

Abstract

N-glycosylation plays essential roles in the folding, trafficking, and maturation of proteins in the secretory pathways, but how individual protein- and site- specific 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 and optimized a magnetic polyethyleneimine boronic acid-containing scaffold (mPBA) enrichment workflow to achieve sensitive and broad enrichment of intact glycopeptides for mass spectrometry analysis, requiring only 0.1 to 0.5 mg total peptide input. With this method, we performed a large intact glycopeptide comparative study, systematically analyzing 13,759 unique protein-, site-, and glycoform combinations, termed glycopeptidoforms, in normal and stressed human cells. The data reveals a dynamic rewiring of N-glycosylation involving hundreds of proteins with complex protein-, site-, and glycan- specific granularity. The magnitude of differential glycosylation far exceeds that of protein expression changes. Individual glycoform reconfigurations can be observed that indicate likely disruptions within specific steps in protein maturation and trafficking. Mannose trimming emerges as a shared disruption across multiple proteins, suggesting a processing bottleneck of the ER stress glycoproteome. 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. The data can be visualized on https://glycoproteome.info.