Characterization of Cytokine Treatment on Human Pancreatic Islets by Top‐Down Proteomics

Type 1 diabetes (T1D) results from autoimmune-mediated destruction of insulin-producing β cells in the pancreatic islet. This process is modulated by pro-inflammatory cytokine signaling, which has been previously shown to alter protein expression in ex vivo islets. Herein, we applied top-down proteomics to globally evaluate proteoforms from human islets treated with proinflammatory cytokines (interferon-γ and interleukin-1β). We measured 1636 unique proteoforms across six donors and two time points (control and 24 h post-treatment) and observed consistent changes in abundance across the glicentin-related pancreatic polypeptide (GRPP) and major proglucagon fragment regions of glucagon, as well as the LF-19/catestatin and vasostatin-1/2 region of chromogranin-A. We also observe several proteoforms that increase after cytokine-treatment or are exclusively observed after cytokine-treatment, including forms of beta-2 microglobulin (B2M), high-mobility group N2 protein (HMGN2), and chemokine (C-X-C motif) ligands (CXCL). Together, our quantitative results provide a baseline proteoform profile for human islets and identify several proteoforms that may serve as interesting candidate markers for T1D progression or therapeutic intervention. SUMMARY: This work applies a top-down proteomics workflow for the characterization and label-free quantification of proteoforms from human islets in the context of inflammation. The workflow is optimized for challenges unique to the islet proteome including high disulfide-linkage content and frequent truncation events, resulting in many proteoforms < 5kDa. There are limited examples of top-down proteomics characterization of human islets, thus this study provides a baseline characterization of the proteoforms of major hormones including chromogranin-A (CHGA), chromogranin-B/ secretogranin-1 (CHGB/SCG1), chromogranin-C/ secretogranin-2 (CHGC/SCG2), islet amyloid polypeptide (amylin/IAPP), insulin (INS), glucagon (GCG), pancreatic polypeptide prohormone (PPY), somatostatin (SST), and neurosecretory protein VGF (VGF). The quantitative results of proteoform abundances before and after cytokine treatment, which mimics the proinflammatory environment during T1D progression, provides interesting insights on how prohormone processing is altered under a proinflammatory environment.