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,Kym F Faull

doi:10.1194/jlr.m500188-jlr200

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.

Highlights

A mass spectrometric method is described for monitoring cerebrosides in the presence of excess concentrations of alkali metal salts
The possibility of following sulfatide depletion by electrospray ionization mass spectrometry (ESI) has been tested by taking advantage of the strong signals that these lipids yield in the negative ion mode [16, 17]; more importantly, the possibility of following cerebroside formation has been tested by taking advantage of the strong signals that these lipids yield as their lithiated adducts in the positive ion mode [18]. These two approaches have been tried, and here we present the results of this comparison, which showed that monitoring cerebroside formation by ESI coupled to tandem mass spectrometry (MS/MS) provides a reliable, fast, specific, nonradioactive method for assays involving both cerebroside sulfate activator protein (CSAct)- and detergent-solubilized sulfatide
The negative ion ESI spectra of brain sulfatide reveal a series of ions from m/z 806 to 950, reflecting the relative abundance of the various species of this lipid in the brain (Fig. 4A) [16, 17]

Summary

Chemicals and reagents

Bovine brain sulfatide and cerebroside and semisynthetic [2H35] stearoyl cerebroside ([2H35]C18:0 cerebroside) were obtained from Matreya, Inc. (State College, PA). Bovine brain sulfatide and cerebroside and semisynthetic [2H35] stearoyl cerebroside ([2H35]C18:0 cerebroside) were obtained from Matreya, Inc. Taurodeoxycholate, lithium chloride, and BSA were from Sigma Aldrich (St. Louis, MO). Quartzdistilled water (Ͻ16 m⍀/cm) was produced in house, and all other reagents and solvents were of analytical grade or better

Preparation of synthetic sulfatides

Data processing

RESULTS AND DISCUSSION