PNNL

Abstract

Aims: This work aimed to explore the role of multiple types of protein thiol post-translational modifications (PTMs) in pancreatic ß-cells under endoplasmic reticulum (ER) stress through establishing a direct detection workflow. Results: The LC-MS/MS based workflow allows simultaneous quantification of protein abundances and multiple types of thiol PTMs. The application to mouse pancreatic ß-cells (Beta-TC-6) with thapsigargin-induced ER stress resulted in the quantification of > 9000 proteins and eight types of thiol PTMs, including intra-peptide disulfide (S-S), S-glutathionylation (SSG), S-sulfenylation (SOH), S-sulfinylation (SO2H), S-sulfonylation (SO3H), S-nitrosylation (SNO), and S-polysulfidation (persulfidation, SSH and trisulfidation, SSSH). Significant changes of protein abundances involved in canonical pathways such as autophagy, unfolded protein response, protein ubiquitination pathway, and EIF2 signaling were observed. Moreover, ~1,700 Cys sites were observed with one or multiple types of PTMs with SSH and S-S as the predominant types of modifications. Significant changes in the levels of different PTMs were also observed on various enzymes and their active sites, suggesting distinctive translational and post-translational regulation of enzyme activity. The observed complexity of thiol modifications on the same Cys residues illustrate the challenge in the characterization and interpretation of protein thiol modifications in biology. Innovation: The direct detection workflow preserves thiol PTMs and allows profiling of different types of thiol PTMs concurrently in a single experiment. Conclusion: Simultaneous global proteome and thiol PTM profiling was enabled by the direct detection workflow. The data revealed the complexity of thiol redox PTMs and their regulation in pancreatic beta cells under ER stress.