High mobility group box-1 (HMGB1) is a non-histone chromosomal protein that is highly conserved in eukaryotic cells. It is known to play a regulatory role in inflammatory immune responses and has recently proved to be a potential novel therapeutic target in malignant mesothelioma (MM). HMGB1 normally locates in the nucleus. However, during cell necrosis due to asbestos fibers, HMGB1 undergoes acetylation followed by translocation from the nucleus to the cytoplasm, and then secreted to extracellular space, where it binds to and activates pro-inflammatory mediators. Given the role it plays in inflammatory processes, HMGB1 may hold promise as a biomarker of cell transformative processes and thus hold utility as an indicator of asbestos exposure. A recent report reveals that the serum levels of both HMGB1 and acetylated HMGB1 are elevated in MM patients using an HMBG1 ELISA kit. Herein, we developed a stable isotope dilution HPLC-MS method, which has higher sensitivity and specificity compared with currently available HMBG1 ELISAs, to accurately quantify the HMGB1 levels and the acetylated HMGB1 levels in serum. Stable isotopically labeled HMGB1 was expressed using Stable isotope labeling by amino acids in cell culture (SILAC) strategy and was added as the internal standard (ISTD) at the initial step of the sample preparation. An anti-acetylation antibody was utilized to immunoprecipitate acetylated HMGB1 from the serum. In addition, dimethyl pimilimidate (DMP) was used to cross-link the antibody with the magnetic beads, in order to enable the elution of the target. Following elution, Glu-C digestion of HMBG1 yields peptides including two nucleus localization signal (NLS) fragments. These two key peptides are highly acetylated, which prevents HMGB1 from reentering the nucleus. Absolute quantification was achieved by analyzing the ratio of these two peptides from endogenous form and ISTD. The serum actylated HMGB1 levels in mesothelioma patiens were compared with healthy controls as well as with individuals that were heavily exposed to asbestos. The improved accuracy and enhanced sensitivity of this assay provides a thorough quantification method for acetylated HMGB1 and further identifies if the acetylated form is a promising biomarker for mesothelioma patients.

Accurate quantification of serum protein mesothelioma biomarkers

254th National Meeting (2017)

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