Increased oxidized ribose, glucose, glyoxal and methylglyoxal concentrations are observed in several metabolic diseases including diabetes and metabolic syndrome, mediating the non-enzymatic protein glycation and carbonylation followed by protein unfolding, cross-linking and aggregation.  In this presented research, we hypothesized that the protein posttranslational modifications induced by glycation and glycoxidation, i.e. the advanced glycation end products (AGE), will impair the immunological responses in mice model of T2DM metabolic syndrome.  To test our hypothesis, we analyzed the AGE-modified proteomes and the immunopeptidomes eluted from MHC-II receptors, using the CD11c+ purified primary dendritic cells from healthy, controls and <i>Ob/Ob</i> mice models of T2DM.  The peptides bearing AGE-amide adducts were sequenced by bottom-up label-free MS/MS by employing a QExactive HF quadrupole mass spectrometer for global proteomics profiling.  The immunopeptidomes were sequenced using the top-down MS/MS using an Orbitrap Tribid Fusion mass spectrometer by employing a combination of data-dependent (DDA) and data independent acquisition (DIA) methodologies.  Bioinformatics analysis highlighted the several cellular pathways affected by AGE, including endosomal maturation and antigen presentation and processing machinery.  Analysis of the type of AGE-PTM accumulated in the proteomes from Ob/Ob samples ranked the site-specific carboxymethylation of lysine (CML) as the most abundant AGE found in the glycated proteome.  Further mapping of PTM on the <i>in vitro</i> glycated human recombinant MHC II allowed the identification of key AGE-modified amino acids residues involved in peptide epitope binding, as shown by fluorescence polarization assays.  Overall, these results highlight the potential role for accumulation of AGE-PTM in the pathophysiology of immune responses.

NanoLC/MS/MS profiling of advanced glycation end products (AGE) PTM on MHC-II molecules and components of antigen processing pathway in diabetes

2019 Fall National Meeting

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