Simultaneous proteomics and three PTMomics characterization of pro-inflammatory cytokines stimulated INS-1E cells using TiO2 enrichment strategy

Diverse protein post-translational modifications (PTMs) in proteins form complex combinatorial patterns to regulate the protein function and biological processes in a fine-tuning manner. Reversible phosphorylation, cysteines (Cys) modification, and N-linked glycosylation are essentially involved in cellular signaling pathways of pro-inflammatory cytokines, which can induce beta cell death and diabetes. Here we developed a novel mass spectrometry-based proteomic strategy (termed TiCPG) for the simultaneous comprehensive characterization of the proteome and three post-translational modifications (PTMomes) by applying TiO2 enrichment of peptides with reversibly modified Cysteine (rmCys), Phosphorylation, and sialylated N-linked (SAN-) Glycosylation from low amount of sample material with largely minimized sample loss. We applied this TiCPG strategy to quantitatively study the change of the three PTMs in {beta}-cell-like INS-1E cells subject to pro-inflammatory cytokines stimulation. It enabled efficient enrichment and quantitative analysis of 8346 rmCys sites, 10321 phosphosites and 1906 SAN-glycosylation sites from 5853 proteins. Significant regulation was found on 100 proteins at the total protein level, while much higher degree of regulation was identified on 3025 peptides with PTMs from 1490 proteins. The three PTMs were co-regulated in proteins, but demonstrated differential spatial and temporal patterns related to protein cellular localization and function in the time course of cytokines stimulation, and they were extensively involved in essential signaling pathways related to pro-inflammatory cytokine mediated {beta}-cell apoptosis, such as the inducible NO synthase (NOS2) signaling pathway, Overall, the TiCPG strategy is proved as a straight forward and powerful tool for multiple PTMomics studies.