A sequential (phospho)proteomics enrichment approach reveals distinct protein phosphorylation patterns of mesothelial cells exposed to high glucose containing PD fluid or heat stress

Peritoneal dialysis (PD) is a kidney function replacement therapy, where the peritoneum is used as a semi-permeable membrane to remove water, toxins, and solutes from patients by a glucose-based PD-fluid. Exposure to PD-fluid triggers changes in peritoneal mesothelial cells and has been shown to impair cellular stress response (CSR) mechanisms. Glucose degradation products (GDPs) in the PD-fluids lead to diabetes-like vasculopathy and are speculated to block critical elements of the CSR which is highly regulated via PTMs. This study aims to identify system-wide protein phosphorylation impairing the CSR leading to deterioration of the peritoneal membrane and poor clinical outcome.

Human mesothelial cells exposed to heat, PD-fluids or GDPs were analyzed with a quantitative (phospho)proteomics approach. 68 samples were labeled with TMTpro-18-plex. Phosphopeptides were enriched in a sequential approach, first using TiO2, followed by FeNTA enrichment of the flow-through. The two phospho-peptide enriched eluates were combined and samples were high-pH RP offline-fractionated. Both sample types were analyzed by nano-LC /Orbitrap-Exploris480 with FAIMSpro. An internal pooled standard consisting of equal parts of all samples was used to link multiple TMT-runs. Database search and quantification were performed using Proteome Discoverer.

The approach enabled identification of >10,100 proteins (>9,000 with abundance in all runs), 46,848 phospho-peptides and 38,336 unique phospho-sites. 83% phospho-sites were serine residues, 16% threonines and 1% tyrosines. The analysis of the total proteome showed a shift to biological processes related to chaperone activity and CSR in heat but not in PD-fluid conditions. The quantitative phosphoproteomics approach revealed differentially de/activated players in the HSF-1 activation pathway and its effector kinases depending on the presence of GDPs.

This approach enables high coverage of the total and phosphoproteome from the same sample and thereby enabling analysis of relevant processes of the mesothelial reaction to PD-fluid induced stress possibly leading to identification of new therapeutic targets.