Analysis of site-specific N-homocysteinylation of human serum albumin in vitro and in vivo using MALDI-ToF and LC-MS/MS mass spectrometry.

Elevated levels of homocysteine (Hcy) are associated with cardiovascular and neurodegenerative diseases in humans. Hcy becomes a component of human proteins as a result of N-homocysteinylation of protein lysine residues by Hcy-thiolactone, which affects the protein's structure and function, and contributes to Hcy-related pathology. Albumin is the major target for N-homocysteinylation in human blood in vivo. Previous work has identified Lys-525 as a predominant site of N-homocysteinylation in vitro and in vivo. Here we show that Lys-4, Lys-12, Lys-137, Lys-159, Lys-205, and Lys-212 of human albumin are susceptible to N-homocysteinylation in vitro and provide evidence that two of those residues, Lys-137 and Lys-212, in addition to Lys-525, are N-homocysteinylated in vivo in human plasma.