J. Lipid Res.
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A more recent version of this article appeared on October 1, 2005

Papers In Press, published online ahead of print August 1, 2005
J. Lipid Res., doi:10.1194/jlr.M500188-JLR200
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Submitted on May 10, 2005
Revised on July 19, 2005
Accepted on July 21, 2005

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

Andrew J. Norris, 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

Chemistry & Biochemistry, Pasarow Mass Spectrometry Laboratory, UCLA, Los Angeles, CA 90019

Corresponding Author: 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 aryl sulfatase 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 just 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 CSAct/ASA 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 where the assay could not be previously performed because of the prior need for radio-labeled substrate which is now not required.


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V. E. Ahn, P. Leyko, J.-R. Alattia, L. Chen, and G. G. Prive
Crystal structures of saposins A and C
Protein Sci., August 1, 2006; 15(8): 1849 - 1857.
[Abstract] [Full Text] [PDF]


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