Label free DIA and DDA nano‐LC/MS/MS improved quantitative profiling of redox stress mediated proteomic changes in mouse dendritic cells

Abstract

A diet rich in saturated fat and carbohydrates causes a low‐grade chronic inflammation in several organs. To investigate the impact of oxidative stress and metabolic syndrome on the whole cellular proteome of professional antigen presenting cells (APCs), we isolated six biological replicates consisted of total proteomic extracts of dendritic cells (DCs) from B6 mice on normal and high fructose/high fat (HFHF) diet and performed quantitative label‐free data independent (DIA) and data dependent (DDA) nano LC‐MS/MS analysis of protein expression profiles. Our optimized primary DCs processing for label free proteomics and DIA quantitative analysis retrieved 1451 proteins (with at least 1 unique peptide), identified at 2% attained FDR (0.5% FDR for peptides) out of which 744 proteins were eligible for quantitative analysis and exhibited statistically significant changes in mouse DCs proteomes harvested from mice on HFHF vs normal diet (ANOVA, p<0.05). Bioinformatics and GO annotations mapped quantitative changes in energy and stress related proteins associated with several metabolic pathways including glycolysis, lipolysis and mitochondrial oxidative phosphorylation, reflecting the possible onset of insulin‐resistance phenotype. The (LFQ) DIA analysis highlighted qualitative and quantitative changes in the proteomes from mouse DCs, spanning a wide spectrum of up‐regulated inflammatory pathways, which in turn, could wire the reshaping of immunopeptidome landscape presented by MHC‐II under redox metabolic stress.