Kinetics of albumin homocysteinylation measured with matrix-assisted laser/desorption ionization mass spectrometry versus with a radioactive tracer.

Homocysteinylation is a post-translational protein modification which involves homocysteine-thiolactone and may be responsible for many pathophysiological changes secondary to hyperhomocysteinemia. Therefore, methods to measure protein homocysteinylation in intact biological samples are required. We tested whether matrix assisted-laser/desorption ionization mass spectrometry (MALDI-MS) can detect time- and dose-dependent changes in in vitro homocysteine-thiolactone binding to human serum albumin. We have compared this method with a 35S-thiolactone radioactive binding assay. Incubations with and without dithiothreitol allowed measurement of the amide-linked and disulfide-linked thiolactone-protein adducts, respectively. A good correspondence in time- and dose-dependent protein-thiolactone formation was observed between the two methods. A maximum of 9 to 12 thiolactone residues were bound to each albumin molecule. The 35S-thiolactone bound albumin tightly, particularly at the lowest concentrations, with approximately 70% of the binding amide-linked. Although the results of the two methods were rather similar, the radioactive method appears to be more sensitive than the MALDI-MS technique.