Scheme 2.

Procedure for preparation and redox analysis of secreted HMGB1. A549 cell culture media samples are incubated with iodoacetamide (IAA) to carbamidomethylate free (reduced) Cys residues. tris(2-carboxyethyl)-phosphine (TCEP)-reduced (SILAC) HMGB1 is added both before and after the reaction to act as an internal standard for both free (reduced) and disulfide (oxidized) HMGB1. HMGB1 is immunoprecipitated, eluted, and reduced with TCEP prior to N-ethylmaleimide derivatization. Tryptic peptides for PRM analysis are prepared by overnight trypsin digestion, whereas Glu-C peptides are prepared by overnight Glu-C digestion following in-solution CD₃-acetylation of the lysine residues with D6-acetic anhydride.