Clinical studies have shown that elevated plasma total Hcy is associated with an increased risk of the development of vascular and neurological diseases. An underlying mechanism of Hcy pathogenicity may involve Hcy incorporation into protein, which occurs in the human body. The pathway involves metabolic conversion of Hcy to Hcy-thiolactone catalyzed by methionyl-tRNA synthetase during protein biosynthesis. Hcy-thiolactone is chemically reactive and forms adducts with protein, N-Hcy-protein, in which the carboxyl group of Hcy is linked by an amide bond to ε-amino group of a protein lysine residue. This reaction impairs protein structure and function. Major pathophysiological consequences of protein N-homocysteinylation include induction of anti-N-Hcy-protein auto-antibodies and thrombogenesis in humans, which contribute to atherosclerosis in humans. The bulk of Hcy circulating in human blood is N-linked to hemoglobin and albumin. We have previously reported that Lys525 is a predominant site of N-homocysteinylation in human serum albumin in vitro and in vivo.. In the present work we analyzed native and Hcy-thiolactone-modified human serum proteins by proteomic approaches. Protein samples were digested with trypsin; resulting peptides were purified by HPLC and subjected to MALDI-TOF mass spectrometric analyses. We found that Lys4, Lys12, Lys137, Lys159, and Lys99 are sites for the modification of human serum albumin by Hcy-thiolactone. Two sites in hemoglobin susceptible to the modification of by Hcy-thiolactone, βLys18 and αLys16, were also identified. Furthermore, we have found that a peptide containing N-Hcy-Lys525 is easily detected in MALDI-TOF mass spectrum of a tryptic digest of human serum proteins. Based on this finding, we have developed a method that allows direct monitoring of N-Hcy-albumin in human serum.

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