title: Quantification of cellular acid sphingomyelinase and galactocerebroside beta-galactosidase activities by electrospray ionization mass spectrometry.
abstract: BACKGROUND
  Diagnosis of Niemann-Pick (A and B) and Krabbe diseases is achieved by measurement of the lysosomal enzymes acid sphingomyelinase (ASM) and galactocerebroside beta-galactosidase (GCG), respectively. Conventional assays use radiolabeled or fluorescent substrates and do not allow simultaneous determination of two or more enzymes in the sample.
  
  
  METHODS
  We developed a sensitive and specific method to assay ASM and GCG in skin fibroblast homogenates using biotinylated substrate conjugates. The products were purified by bioaffinity capture on streptavidin-agarose beads and, following release, were analyzed by electrospray ionization mass spectrometry. Quantification was achieved using stable-isotope-labeled internal standards that were chemically identical to the products of the enzymatic reactions.
  
  
  RESULTS
  The method demonstrated excellent linearity of ASM and GCG enzymatic product formation with the amount of cellular protein and incubation time. The range of ASM activities in fibroblast lysates from six healthy patients was 39-70 nmol. mg(-1). h(-1) compared with 3.7-5.1 nmol. mg(-1). h(-1) in cell lysates from two patients affected with Niemann-Pick A disease. The GCG activities toward the corresponding substrate conjugate were 4.0-6.8 nmol. mg(-1). h(-1) in cell lysates from healthy patients compared with 0.1-0.2 nmol. mg(-1). h(-1) in cell lysates from two patients affected with Krabbe disease. The amounts of substrate conjugates needed per analysis were 15 nmol (14 microg) for both ASM and GCG.
  
  
  CONCLUSIONS
  Electrospray mass spectrometry combined with the use of biotinylated substrate conjugates and bioaffinity purification represents a new approach for the diagnosis of lysosomal storage diseases as demonstrated for Niemann-Pick A and Krabbe diseases. No radioactive substrates are used, and the method uses a single instrumental platform to determine both ASM and GCG in one cell sample.
authors.affiliation: Department of Chemistry, University of Washington, Seattle, WA 98195-1700, USA.
authors.forename: X
authors.initials: X
authors.lastname: Zhou
authors.forename: F
authors.initials: F
authors.lastname: Turecek
authors.forename: C R
authors.initials: CR
authors.lastname: Scott
authors.forename: M H
authors.initials: MH
authors.lastname: Gelb
mesh_terms: D019921:Biotinylation; D002478:Cells, Cultured; D005347:Fibroblasts / Q000737:chemistry; D005698:Galactosylceramidase / Q000032:analysis*; D006801:Humans; D007965:Leukodystrophy, Globoid Cell / Q000175:diagnosis*; D009682:Magnetic Resonance Spectroscopy; D009542:Niemann-Pick Diseases / Q000175:diagnosis*; D012680:Sensitivity and Specificity; D012867:Skin / Q000166:cytology; D021241:Spectrometry, Mass, Electrospray Ionization; D013108:Sphingomyelin Phosphodiesterase / Q000032:analysis*
chemical_list: D013108:Sphingomyelin Phosphodiesterase; D005698:Galactosylceramidase