Simultaneous proteomics and three PTMomics characterization of pro-inflammatory cytokines stimulated INS-1E cells using TiO₂enrichment strategy

Abstract

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 TiO 2 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 β -cell-like INS-1E cells subject to pro-inflammatory cytokines stimulation. It enabled efficient enrichment and quantitative analysis of 8346 rmCys sites, 10321phosphosites 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 β -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. signal transducer and activator of transcription proteins; NF-κB, nuclear factor-kappa B; NOS2, inducible NO synthase;diabetes [14]. The oxidative status of Cys, phosphorylation and glycosylation could be significantly regulated in this process, but it is largely unknown on a global scale. Therefore, in this study, we combined the three TiO 2 enrichment methods to develop a comprehensive TiCPG strategy for simultaneous analysis of the proteome and the three abovementioned PTMomes from low amount of starting material, and applied this multiple PTMomics strategy to quantitatively study the change of proteome and these three PTMomes, and their interaction in insulinoma pancreatic beta-cell line, INS-1E cells subject to IFN-γ/TNF-α co-stimulation using iTRAQ and LC-MS/MS based quantitative proteomics. Results and DiscussionThe TiO 2 -based simultaneous enrichment of reversibly modified Cys peptides, Phosphopeptides, and sialylated N-linked Glycopeptides (TiCPG) strategy Diverse PTMs form complex combinatorial patterns and cooperate to specify downstream biological processes in a fine-tuning manner. The combined characterization of different PTMs within the same sample is highly desired for systemically analyzing PTMs in biolog...

Protein Oxidative Modifications in Neurodegenerative Diseases: From Advances in Detection and Modelling to Their Use as Disease Biomarkers

“…However, recent technological advances, particularly in computational simulations, have facilitated the development of new strategies to study this complex interplay of PTMs [157]. In addition, new MS-based methodologies, coined PTMomics, have been developed in recent years to identify multiple PTMs simultaneously, including phosphorylation, cysteine modifications, and N-linked glycosylation [158,159]. For instance, this approach has successfully been used in the context of PD neurons in culture, uncovering the role of various PTMs in the pathophysiology of PD [160].…”

A novel post-translational proteomics platform identifies neurite outgrowth impairments in Parkinson’s diseaseGBA-N370Sdopamine neurons

“…Applying a novel multi-part enrichment strategy (TiCPG) (1), we have isolated and quantified peptides with phosphorylation, reversible cysteine-modification or sialylated N-linked glycosylation, simultaneously. We applied this methodology to iPSC-derived dopamine neurons from GBA - N370S PD patients and healthy age-matched controls, identifying large numbers of dysregulated native and modified proteins.…”