TiCPG-a strategy for the simultaneous enrichment of reversibly modified cysteine peptides, phosphopeptides, and sialylated N-Glycopeptides to study cytokines stimulated beta-cells

Diverse post-translational modifications (PTMs) regulate protein function and interaction to fine-tune biological processes. Reversible phosphorylation, cysteines (Cys) modifications, and N-linked glycosylation are all essen-tially involved in cellular signaling pathways, such as those initiated by the action of pro-inflammatory cyto-kines, which can induce pancreatic ll-cell death and diabetes. Here we have developed a novel strategy for the simultaneous and comprehensive characterization of the proteome and three PTMs including reversibly modified Cysteines (rmCys), phosphorylation, and sialylated N-linked glycosylation from low amount of sample material. This strategy, termed TiCPG, is based on a combination of chemical labeling and titanium dioxide (TiO2) chromatography. We applied the TiCPG strategy to study the proteome and the three PTMs changes in ll-cells subject to pro-inflammatory cytokines stimulation. It enabled quantitative analysis of 8346 rmCys sites, 10,321 phosphosites and 962 sialylated N-glycosites from 5496 proteins. Significant regulation was found on 100 proteins at the expression level, while 3020 PTM peptide isoforms from 1468 proteins were significantly regu-lated. The three PTMs were involved in cytokine mediated ll-cell apoptosis, such as the NFxB and the inducible NO synthase signaling pathways. Overall, the TiCPG strategy is a cheap, straightforward, and powerful tool for studies targeting the three PTMs described above.Significance: The present study presents a fast and easy method for quantitative assessment of the proteome and three PTMs from minimal amount of sample material. This simple method provides comprehensive and signif-icant knowledge on biological systems and cellular signaling with relatively low analysis time, suitable for younger researchers and researchers that do not have direct access to LC-MSMS in their laboratories. From sub-milligram amount of material, we were able to map known cellular signaling events of proinflammatory cytokine effect on beta-cells and to discover novel PTMs involved in several known signaling pathways.

Automated summary

A novel strategy called TiCPG has been developed for comprehensive characterization of the proteome and three post-translational modifications (PTMs) including reversibly modified cysteines, phosphorylation, and sialylated N-linked glycosylation. This strategy combines chemical labeling and titanium dioxide chromatography. The application of TiCPG to pro-inflammatory cytokine-stimulated ll-cells revealed significant regulation of protein expression and PTMs, highlighting the involvement of these PTMs in cell apoptosis.