Date of Award
Doctor of Philosophy (PhD)
Ian A. Blair
High mobility group box 1 (HMGB1) is a protein that is released in several pathological contexts, including acetaminophen (APAP) overdose, and modulates the subsequent immune response to tissue damage. Oxidation of HMGB1 potentially regulates its interactions with the immune system but there have been a number of contradictory studies on the redox behavior of HMGB1 and what proteoforms of HMGB1 are present in oxidative pathology. Therefore, a stable isotope dilution two-dimensional nano-ultrahigh-performance liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry (2D-nano-UHPLC-PRM/HRMS) method was developed along with a hepatocarcinoma cell model of APAP overdose to allow for characterization of HMGB1 redox behavior during APAP toxicity. Oxidative modifications to cysteine (Cys) residues (Cys-23, Cys-45, and Cys-106) that are present in HMGB1 were characterized and quantified using carbamidoethyl (CAE) derivatization before and after reduction as well as by direct analysis of disulfide cross-linked peptides. A stable isotope labeled form of HMGB1 was used as an internal standard to correct for sample-to-sample differences in immunoaffinity precipitation (IP), derivatization, and electrospray ionization. The metabolism, toxicity and release of HMGB1 in response to APAP were characterized in a cell model of APAP overdose. Four discreet HMGB1 proteoforms were found to be released from cells following a 24-hour exposure to toxic levels of APAP. Reduced HMGB1 with all three Cys-residues in their free thiol state accounted for 18% of the secreted HMGB1. The proteoform with disulfide between Cys-23 and Cys-45 accounted for 24% of the HMGB1. No evidence was obtained for a disulfide cross-link between Cys-106 and the other two Cys-residues. However, 45% of the HMGB1 formed a cross-link with unidentified intracellular proteins via an intermolecular disulfide bond and 12% was present as cysteic acid. Secreted plasma HMGB1 Cys-23/Cys45 disulfide proteoform together with the Cys-106/protein disulfide proteoforms could potentially serve as early biomarkers of hepatoxicity after APAP overdose as well as biomarkers of drug-induced liver injury (DILI).
Pirnie, Ross, "Characterizing And Quantifying The Redox Behavior Of Cytokine Hmgb1 In Drug-Induced Liver Injury Via High Resolution Mass Spectrometry" (2022). Publicly Accessible Penn Dissertations. 5235.