title: A novel mass spectrometric assay for the cerebroside sulfate activator protein (saposin B) and arylsulfatase A.
abstract: 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 non-hydroxylated 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.
authors.affiliation: Pasarow Mass Spectrometry Laboratory, Department of Psychiatry, and Neuropsychiatric Institute, University of California Los Angeles, Los Angeles, CA 90024, USA. anorris@mednet.ucla.edu
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Norris
authors.forename: Julian P
authors.initials: JP
authors.lastname: Whitelegge
authors.forename: Arman
authors.initials: A
authors.lastname: Yaghoubian
authors.forename: Jean-Rene
authors.initials: JR
authors.lastname: Alattia
authors.forename: Gilbert G
authors.initials: GG
authors.lastname: Privé
authors.forename: Tatsushi
authors.initials: T
authors.lastname: Toyokuni
authors.forename: Hubert
authors.initials: H
authors.lastname: Sun
authors.forename: Mai N
authors.initials: MN
authors.lastname: Brooks
authors.forename: Luigi
authors.initials: L
authors.lastname: Panza
authors.forename: Pamela
authors.initials: P
authors.lastname: Matto
authors.forename: Federica
authors.initials: F
authors.lastname: Compostella
authors.forename: Natascha
authors.initials: N
authors.lastname: Remmel
authors.forename: Ralf
authors.initials: R
authors.lastname: Klingenstein
authors.forename: Konrad
authors.initials: K
authors.lastname: Sandhoff
authors.forename: Claire
authors.initials: C
authors.lastname: Fluharty
authors.forename: Arvan
authors.initials: A
authors.lastname: Fluharty
authors.forename: Kym F
authors.initials: KF
authors.lastname: Faull
mesh_terms: D000818:Animals; D002417:Cattle; D002553:Cerebroside-Sulfatase / Q000032:analysis*; D008094:Lithium / Q000737:chemistry; D049231:Saposins / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D049231:Saposins; D008094:Lithium; D002553:Cerebroside-Sulfatase