A novel mass spectrometric assay for the cerebroside sulfate activator protein (saposin B) and arylsulfatase A

A mass spectrometric method is described for monitoring cerebrosides in the presence of excess concen- trations of alkali metal salts. This method has been adapted for use in the assay of arylsulfatase A (ASA) and the cere- broside sulfate activator protein (CSAct or saposin B). De- tection of the neutral glycosphingolipid cerebroside prod- uct was achieved via enhancement of ionization efficiency in the presence of lithium ions. Assay samples were ex- tracted into the chloroform phase as for the existing assays, dried, and diluted in methanol-chloroform-containing lith- ium chloride. Samples were analyzed by electrospray ioniza- tion mass spectrometry with a triple quadrupole mass spec- trometer in the multiple reaction monitoring tandem mass spectrometric mode. The assay has been used to demon- strate 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 as- say could not be performed previously because of the need for radiolabeled substrate, which is now not required. — Norris, A. J., J. P. Whitelegge, A. Yaghoubian, J-R. Alattia, G. G. Prive, T. Toyokuni, H. Sun, M. N. Brooks, L. Panza, P. Matto, F. Compostella, N. Remmel, R. Klingenstein, K. Sandhoff, C. Fluharty, A. Fluharty, and K. F. Faull. A novel mass spectrometric assay for the cerebroside sulfate activa- tor protein (saposin B) and arylsulfatase A. J. Lipid Res. 2005. 46: 2254-2264.