BACKGROUND: Treatments are being developed for an increasing number of mucopolysaccharidoses, and early diagnosis is expected to be necessary to maximize the benefits of therapy. Therefore, we developed an assay for N-acetylgalactosamine-6-sulfate sulfatase (GALNS), the enzyme deficient in mucopolysaccharidosis IVA (Morquio A syndrome), that is applicable for clinical diagnosis. METHODS: A novel substrate for GALNS was synthesized for a new enzyme activity assay that is based on tandem mass spectrometry and uses dried blood spots (DBSs) as the enzyme source. We optimized the assay conditions, including the substrate concentration, reaction pH, lead formate concentration, incubation time, punch size of the DBS, and mass spectrometer conditions. We also assessed inter- and intraassay variation. RESULTS: The assay uses either solid-phase or liquid-phase extraction before analysis by mass spectrometry. An evaluation of blood spots from 90 randomly chosen healthy newborns and 9 patients with Morquio A syndrome showed a well-defined interval between their respective enzyme activities. Inter- and intraassay imprecision was <10%. CONCLUSIONS: This tandem mass spectrometry assay requires a minimal number of sample-preparation steps, thus making it easy to implement. The assay has the potential to be adopted for early diagnosis of Morquio A syndrome. We believe this assay could be performed in a multiplex fashion with assays for other lysosomal enzymes.
BACKGROUND: Treatments are being developed for an increasing number of mucopolysaccharidoses, and early diagnosis is expected to be necessary to maximize the benefits of therapy. Therefore, we developed an assay for N-acetylgalactosamine-6-sulfate sulfatase (GALNS), the enzyme deficient in mucopolysaccharidosis IVA (Morquio A syndrome), that is applicable for clinical diagnosis. METHODS: A novel substrate for GALNS was synthesized for a new enzyme activity assay that is based on tandem mass spectrometry and uses dried blood spots (DBSs) as the enzyme source. We optimized the assay conditions, including the substrate concentration, reaction pH, lead formate concentration, incubation time, punch size of the DBS, and mass spectrometer conditions. We also assessed inter- and intraassay variation. RESULTS: The assay uses either solid-phase or liquid-phase extraction before analysis by mass spectrometry. An evaluation of blood spots from 90 randomly chosen healthy newborns and 9 patients with Morquio A syndrome showed a well-defined interval between their respective enzyme activities. Inter- and intraassay imprecision was <10%. CONCLUSIONS: This tandem mass spectrometry assay requires a minimal number of sample-preparation steps, thus making it easy to implement. The assay has the potential to be adopted for early diagnosis of Morquio A syndrome. We believe this assay could be performed in a multiplex fashion with assays for other lysosomal enzymes.
Authors: Yijun Li; C Ronald Scott; Nestor A Chamoles; Ahmad Ghavami; B Mario Pinto; Frantisek Turecek; Michael H Gelb Journal: Clin Chem Date: 2004-08-03 Impact factor: 8.327
Authors: O P van Diggelen; H Zhao; W J Kleijer; H C Janse; B J Poorthuis; J van Pelt; J P Kamerling; H Galjaard Journal: Clin Chim Acta Date: 1990-02-28 Impact factor: 3.786
Authors: Trisha A Duffey; Tanvir Khaliq; C Ronald Scott; Frantisek Turecek; Michael H Gelb Journal: Bioorg Med Chem Lett Date: 2010-08-25 Impact factor: 2.823
Authors: Brian J Wolfe; Farideh Ghomashchi; Tim Kim; Cynthia A Abam; Martin Sadilek; Rhona Jack; Jerry N Thompson; C Ronald Scott; Michael H Gelb; Frantisek Turecek Journal: Bioconjug Chem Date: 2012-03-09 Impact factor: 4.774
Authors: Brian J Wolfe; Sophie Blanchard; Martin Sadilek; C Ronald Scott; Frantisek Turecek; Michael H Gelb Journal: Anal Chem Date: 2010-12-30 Impact factor: 6.986
Authors: C J Hendriksz; P Harmatz; M Beck; S Jones; T Wood; R Lachman; C G Gravance; T Orii; S Tomatsu Journal: Mol Genet Metab Date: 2013-04-10 Impact factor: 4.797