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Journal of Lipid Research, Vol. 46, 2254-2264, October 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

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

Andrew J. Norris^1,*,, Julian P. Whitelegge^*, Arman Yaghoubian^*, Jean-Rene Alattia, Gilbert G. Privé, Tatsushi Toyokuni^**, Hubert Sun^*, Mai N. Brooks, Luigi Panza, Pamela Matto, Federica Compostella, Natascha Remmel^***, Ralf Klingenstein^***, Konrad Sandhoff^***, Claire Fluharty, Arvan Fluharty and Kym F. Faull^*

^* Pasarow Mass Spectrometry Laboratory, Departments of Psychiatry & Biobehavioral Sciences and Chemistry & Biochemistry, and Neuropsychiatric Institute, University of California Los Angeles, Los Angeles, CA 90024
Mental Retardation Research Center, University of California Los Angeles, Los Angeles, CA 90024
^** Department of Molecular & Medical Pharmacology, University of California Los Angeles, Los Angeles, CA 90024
Johnson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, CA 90024
Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, M5G 2M9 Canada
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche, e Farmacologiche, Università del Piemonte Orientale, 28100 Novara, Italy
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università di Milano, 20133 Milano, Italy
^*** Kekule Institut fur Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universitaet Bonn, 53121 Bonn, Germany

Published, JLR Papers in Press, August 1, 2005. DOI 10.1194/jlr.M500188-JLR200

¹ To whom correspondence should be addressed. e-mail: anorris{at}mednet.ucla.edu

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.

Abbreviations: ASA, arylsulfatase A; CSAct, cerebroside sulfate activator protein; ESI, electrospray ionization mass spectrometry; IS, internal standard; MALDI, matrix-assisted laser desorption ionization; MLD, metachromatic leukodystrophy; MRM, multiple reaction monitoring; MS/MS, tandem mass spectrometry; TOF, time-of-flight; U, unit of enzyme activity (the amount of enzyme activity that will catalyze the transformation of 1 µmol of the substrate per minute under standard conditions); UCLA, University of California Los Angeles

Supplementary key words saposins A, B, C, and D • electrospray ionization • tandem mass spectrometry

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