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.
Callegher
Elisabetta
E
Department of Clinical and Experimental Medicine, Division of Metabolic Disease, University of Padova.
Seraglia
Roberta
R
Vettore
Monica
M
Puricelli
Lucia
L
Millioni
Renato
R
Tessari
Paolo
P
D004229:Dithiothreitol; D006710:Homocysteine / Q000031:analogs & derivatives / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D007700:Kinetics; D011868:Radioisotopes / Q000737:chemistry*; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013460:Sulfur Isotopes / Q000737:chemistry; D013997:Time Factors
D011868:Radioisotopes; D012709:Serum Albumin; D013460:Sulfur Isotopes; D006710:Homocysteine; C007957:homocysteine thiolactone; D004229:Dithiothreitol