Abstract |
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.
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Authors | Elisabetta Callegher, Roberta Seraglia, Monica Vettore, Lucia Puricelli, Renato Millioni, Paolo Tessari |
Journal | Rapid communications in mass spectrometry : RCM
(Rapid Commun Mass Spectrom)
Vol. 23
Issue 23
Pg. 3837-42
(Dec 2009)
ISSN: 1097-0231 [Electronic] England |
PMID | 19902417
(Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)
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Copyright | Copyright 2009 John Wiley & Sons, Ltd. |
Chemical References |
- Radioisotopes
- Serum Albumin
- Sulfur Isotopes
- Homocysteine
- homocysteine thiolactone
- Dithiothreitol
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Topics |
- Dithiothreitol
- Homocysteine
(analogs & derivatives, metabolism)
- Humans
- Isotope Labeling
(methods)
- Kinetics
- Radioisotopes
(chemistry)
- Sensitivity and Specificity
- Serum Albumin
(chemistry, metabolism)
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
(methods)
- Sulfur Isotopes
(chemistry)
- Time Factors
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