A mass spectrometric method is described for monitoring cerebrosides in the presence of excess concentrations of alkali metal salts. This method has been adapted for use in the assay of arylsulfatase A ( ASA) and the cerebroside sulfate activator protein ( CSAct or saposin B). Detection of the neutral glycosphingolipid cerebroside product was achieved via enhancement of ionization efficiency in the presence of lithium ions. Assay samples were extracted into the chloroform phase as for the existing assays, dried, and diluted in methanol- chloroform- containing lithium chloride. Samples were analyzed by electrospray ionization mass spectrometry with a triple quadrupole mass spectrometer in the multiple reaction monitoring tandem mass spectrometric mode. The assay has been used to demonstrate several previously unknown or ambiguous aspects of the coupled ASA/ CSAct reaction, including an absolute in vitro preference for CSAct over the other saposins ( A, C, and D) and a preference for the nonhydroxylated species of the sulfatide substrate over the corresponding hydroxylated species. The modified assay for the coupled ASA/ CSAct reaction could find applicability in settings in which the assay could not be performed previously because of the need for radiolabeled substrate, which is now not required.
|Titolo:||A novel mass spectrometric assay for the cerebroside sulfate activator protein (saposin B) and arylsulfatase A|
|Autori interni:||COMPOSTELLA, FEDERICA|
|Parole Chiave:||saposins A, B, C, and D; electrospray ionization; tandem mass spectrometry|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||2005|
|Digital Object Identifier (DOI):||10.1194/jlr.M500188-JLR200|
|Appare nelle tipologie:||01 - Articolo su periodico|