Covalent modification of beta2-microglobulin induced by reactive carbonyl species : high resolution mass spectrometric studies

Beta2-microglobulin (β2m) is responsible for dialysis related amyloidosis (DRA), a destructive osteoarticular disease that affects patients undergoing long-term hemodialysis. Physicochemical and immunochemical analyses showed that one of the constituents of DRA aggregates is an acidic form of β2m, which is generated through covalent modifications induced by reactive carbonyl species (RCS) and glucose. The resulting advanced lipoxidation and glycoxidation end products (ALEs and AGEs) have been detected in long-lived β2m amyloid deposits, mainly by immunoistochemistry.[1] On these evidences we here use high resolution MS to investigate the β2m non-enzymatic glycation and carbonylation, as induced by the main RCS.[2] The identification of post-translational modifications was carried out by direct infusion analysis of the intact protein (top-down proteomics) and NanoLC-ESI-MS/MS analysis of the chymotryptic digest (bottom-up proteomics). After 24 hours of incubation of recombinant β2m with RCS, we are able to identify covalent adducts for dialdehydes (glyoxal and methylglyoxal) occurring on Arg4. Neither cross-links/polymerization events nor adducts for α,β-unsatured compounds were observed. On these bases the optimized methods were used for the same characterization and semi-quantitative analysis of purified and commercial urinary β2m. The obtained results give a deeper insight on the covalently modified β2m and RCS as potential drug targets in DRA and inflammation.