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 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.

Quantification of seminolipid by LC-ESI-MS/MS-multiple reaction monitoring: compensatory levels in Cgt mice

“…SGC is the better studied sulfolipid, as its accumulation in the brain and other tissues due to a genetically inherited defi ciency of arylsulfatase A (ASA) or saposin B (SGC carrier protein) results in metachromatic leukodystrophy, a fatal neurological disease ( 19,20 ). With a suitable internal standard, LC-MS/MS-MRM quantifi cation can be supremely accurate and sensitive, as it can quantify individual sulfolipid isoforms that differ by relatively subtle structural changes in the hydrocarbon chains ( 18,(21)(22)(23) N-labeled forms) because their physical and chemical properties, including extraction effi ciency, chromatographic behavior, and ionization effi ciency, are virtually identical to those of the natural, nonlabeled analyte. Although LC-MS/MS-MRM analysis has recently been used to determine the amounts of seminolipid in saposin A-and prosaposin-defi cient mice ( 23 ), the analyses were performed without an internal standard and relied on absolute signal intensity uncorrected for sample losses during extraction.…”

“…However, SGC is unsuitable as a quantitative standard in the MRM assay for SGG. SGC is an exceptionally stable gas phase ion, requiring stringent conditions for collisionally activated dissociation compared with SGG ( 18,22,38 ). In addition, besides the m/z 97 ion fragment, the collision-induced fragments of SGC are different from those of SGG ( 34 ).…”

Direct tandem mass spectrometric profiling of sulfatides in dry urinary samples for screening of metachromatic leukodystrophy

“…Recently, Meikle's group reported detection and quantitation of urine sulfatides by electrospray ionization mass spectrometry in the negative ion mode [24, 25]. Norris et al [26] have developed an assay for sulfatide detection in brain tissue that was based on positive ion electrospray tandem mass spectrometry of sulfatide-lithium ion adducts. Kuchař et al [27] used tandem mass spectrometry in the negative ion mode to prove massive excretion of urinary sulfatides in patients with function defect of ASA protein activator saposin B (prosaposin and saposin B deficiencies) and also in MLD cases.…”

An efficient and convenient synthesis of deuterium-labelled seminolipid isotopomers and their ESI-MS characterization

“…A mass spectrometry assay based on ESI coupled to tandem mass spectrometry has been developed to monitor sulfatide depletion and cerebroside formation, taking advantage of the strong signals these glycolipids yield both in negative (for sulfatides) and positive (for cerebroside in the presence of lithium) ion mode (Hsu and Turk, 2001;Norris et al, 2005). An extension of this method is now being developed to monitor SGG (negative ion mode) and GG (positive ion mode as the lithiated adduct) concentrations (Faull, K.F., Norris, A. J., Yaghoubian, A., Panza, L., Tanphaichitr, N., Ronchetti, F. et al, unpublished data).…”

“…Severe diseases are associated with defective enzymes in glycolipid metabolism: in humans affected by metachromatic leukodistrophy the deficit of arylsulfatase A, and less frequently the deficit of the lipid transporter saposin B (also known as cerebroside sulfate activator protein), causes the accumulation of sulfatide and subsequent neurological damage (Norris et al, 2005), while in male mice lacking the CGT gene, the absence of GG and SGG may be responsible for apoptosis of germ cells, which subsequently leads to a spermatogenesis arrest at the primary spermatocyte level (Zhang et al, 2005). Since sperm SGG is involved in sperm-egg interaction, it is possible that decreased levels of sperm SGG are one of the causes of male infertility and/or subfertility.…”