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Kinetics of albumin homocysteinylation measured with matrix‐assisted laser/desorption ionization mass spectrometry versus with a radioactive tracer
Author(s) -
Callegher Elisabetta,
Seraglia Roberta,
Vettore Monica,
Puricelli Lucia,
Millioni Renato,
Tessari Paolo
Publication year - 2009
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4290
Subject(s) - chemistry , thiolactone , human serum albumin , mass spectrometry , matrix assisted laser desorption/ionization , albumin , serum albumin , chromatography , biochemistry , stereochemistry , desorption , organic chemistry , adsorption
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 35 S‐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 35 S‐thiolactone bound albumin tightly, particularly at the lowest concentrations, with ≈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. Copyright © 2009 John Wiley & Sons, Ltd.