Mass spectrometry-based direct detection of multiple types of protein thiol modifications in pancreatic beta cells under endoplasmic reticulum stress

Redox Biology

Ronald J. Moore¹  Matthew E. Monroe¹  Vanessa L. Paurus¹  Song Feng¹  Tai-Du Lin¹  Nicholas J. Day¹  Matthew J. Gaffrey¹  Wei-Jun Qian¹  Bin Yang²  Xiaolu Li²  Ming Xian³ 

[1] Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA;Department of Biological Systems Engineering, Washington State University, Richland, WA, 99354, USA;Department of Chemistry, Brown University, Providence, RI, 02912, USA;

关键词: Protein thiols;    Thiol redox modifications;    Direct detection;    Post-translational modifications;    Redox proteomics;   

DOI  :  

来源: DOAJ

【 摘 要 】

Thiol-based post-translational modifications (PTMs) play a key role in redox-dependent regulation and signaling. Functional cysteine (Cys) sites serve as redox switches, regulated through multiple types of PTMs. Herein, we aim to characterize the complexity of thiol PTMs at the proteome level through the establishment of a direct detection workflow. The LC-MS/MS based workflow allows for simultaneous quantification of protein abundances and multiple types of thiol PTMs. To demonstrate its utility, the workflow was applied to mouse pancreatic β-cells (β-TC-6) treated with thapsigargin to induce endoplasmic reticulum (ER) stress. This resulted in the quantification of >9000 proteins and multiple types of thiol PTMs, including intra-peptide disulfide (S–S), S-glutathionylation (SSG), S-sulfinylation (SO2H), S-sulfonylation (SO3H), S-persulfidation (SSH), and S-trisulfidation (SSSH). Proteins with significant changes in abundance were observed to be involved in canonical pathways such as autophagy, unfolded protein response, protein ubiquitination pathway, and EIF2 signaling. Moreover, ~500 Cys sites were observed with one or multiple types of PTMs with SSH and S–S as the predominant types of modifications. In many cases, significant changes in the levels of different PTMs were observed on various enzymes and their active sites, while their protein abundance exhibited little change. These results provide evidence of independent translational and post-translational regulation of enzyme activity. The observed complexity of thiol modifications on the same Cys residues illustrates the challenge in the characterization and interpretation of protein thiol modifications and their functional regulation.

【 授权许可】

Unknown