Advanced glycation end products of beta2-microglobulin in uremic patients as determined by high resolution mass spectrometry

By using a high resolution top-down and bottom-up approach we identified and characterized the AGEs of beta(2)-microglobulin (beta(2)-m) formed by incubating the protein in the presence of glucose and of the main reactive carbonyl species. Glucose induced glycation on the N-terminal residue, while glyoxal (GO) and methylglyoxal (MGO) covalently reacted with Arg3. Carboxymethyl (CM-R) and imidazolinone (R-GO) derivatives were identified in the case of GO and carboxyethyl arginine (CE-R) and methyl-imidazolinone (R-MGO) for MGO. Interestingly, alpha,beta-unsaturated aldehydes [4-hydroxy-2-nonenal (HNE); 4-oxo-2-nonenal (ONE); acrolein (ACR)] did not induce any covalent modifications up to 100 mu M. The different reactivity of beta(2)-m towards the different RCS was then rationalized by molecular modeling studies. The MS method was then applied to fully characterize the AGEs of beta(2)-m isolated from the urine of uremic subjects. CM-R, CE-R and R-MGO were easily identified on Arg3 and their relative abundance in respect to the native protein determined by a semi-quantitative approach. Overall, the AGEs content of urinary psm ranged from 0.2 to 1% in uremic subjects. The results here reported offer novel insights and technical achievements for a potential biological role of AGEs-beta(2)-m in pathological conditions. (C) 2013 Elsevier B.V. All rights reserved.