Abstract 2216: A novel MRM-based mass spectrometry assay to quantify HMGB1
Authors: Chen Dawn Zhaohui , Spivia Weston R. , Gao Yang , Eyk Jennifer Van , Kanodia Shreya 
Published: July 2017

Abstract
Background: High mobility group box 1 (HMGB1) is a versatile protein with dual roles. Within the cell, this highly conserved chromosomal protein functions as a DNA chaperone. Outside of the cell, it functions as the prototypical damage-associated molecular pattern. There is significant evidence that HMGB1 dysfunction contributes to cancer development, particularly in mesothelioma, where its role in carcinogenesis is better defined.
Goal: To develop a mass spectrometry based immune-multiple reaction monitoring (iMRM) assay to quantify HMGB1 and its disease-associated post-translationally modified forms.
Methods: Mesothelioma cell lines (Ren, Phi and PP-Mill) known to secrete HMGB1 were used. Concentrated cell line supernatants, anti-HMGB1 antibody-loaded magnetic beads were incubated overnight. Beads were extensively washed and the HMGB1 released from the beads was enzymatically digested prior to targeted MRM mass spectrometry. Known quantity of N15 labeled synthetic peptides were spiked in to be used as internal standards to assess the total protein concentrations as well as the acetylated modified peptides that encompass the Lys residue. To ensure tight reproducibility during sample processing, we developed an automated workflow from enrichment to digestion with % coefficient of variance of less than 20%.
Results: Two multiplex MRM assays were developed for HMGB1: 1) total HMGB1 protein assay based on trypsin digested peptides and 2) Acetylated HMGB1 assay based on Endoproteinase GluC (Staphylococcus aureus Protease V8) digestion to obtain the peptides containing hyper-acetylated Lysine residues, which are not located within a suitable tryptic peptide. Four significant milestones were accomplished. i) We optimized the HMGB1 liquid chromatography and mass spectrometry performance through the utilization of signature synthesized peptides representing the common amino acid sequence and potential acetylated regions. The calibration curves for both peptides based on the ratio of light and heavy isotopic labeled internal standards were achieved. LLOD and LLOQ and the inter and intra-day variability of the iMRM assay have been achieved. ii) A robust immuno-capture protocol was established. The final protocol included isolation of HMGB1 using a capture antibody followed by protein release, denaturation, reduction, alkylation and digestion. To date, we have optimized the conditions for HMGB1 attachment to antibody, antibody coupling to magnetic beads, the wash and elution steps. iii) The protocol was applied to quantify HMGB1 in mesothelioma cell lines. HMGB1 was measured in cytoplasm, nucleus and supernatant from the cell lines. iv) Finally, the assay was adapted to work on human plasma samples.
Conclusion: We successfully developed the iMRM workflow for quantification of total HMGB1 and acetylated HMGB1 based on the measurement of the target signature peptides in HMGB1.
Citation Format: Dawn Zhaohui Chen, Weston R. Spivia, Yang Gao, Jennifer Van Eyk, Shreya Kanodia. A novel MRM-based mass spectrometry assay to quantify HMGB1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2216. doi:10.1158/1538-7445.AM2017-2216