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. 2010:603:339-50.
doi: 10.1007/978-1-60761-459-3_32.

Multiplex lysosomal enzyme activity assay on dried blood spots using tandem mass spectrometry

X Kate Zhang  1 , Carole S Elbin, Frantisek Turecek, Ronald Scott, Wei-Lien Chuang, Joan M Keutzer, Michael Gelb
Affiliations

Multiplex lysosomal enzyme activity assay on dried blood spots using tandem mass spectrometry

X Kate Zhang et al. Methods Mol Biol. 2010.

Abstract

Deficiencies in any of the 50 degradative enzymes found in lysosomes results in the accumulation of undegraded material and subsequently cellular dysfunction. Early identification of deficiencies before irreversible organ and tissue damages occur leads to better clinical outcomes. In the method which follows, lysosomal alpha-glucosidase, alpha-galactosidase, beta-glucocerebrosidase, acid sphingomyelinase, and galactocerebrosidase are extracted from dried blood spots and incubated individually with an enzyme-specific cocktail containing the corresponding substrate and internal standard. Each enzyme cocktail is prepared using commercially available mixture of substrate and internal standard at the predetermined optimized molar ratio. After incubation, the enzymatic reactions are quenched using an ethyl acetate/methanol solution and all five enzyme solutions are combined. The mixtures of the reaction products are prepared using liquid-liquid and solid-phase extractions and quantified simultaneously using selected ion monitoring on LC-MS-MS system.

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Figures

Fig. 32.1
Fig. 32.1
Substrates, products, and internal standards for the five lysosomal enzyme activity assays. The masses of the products and internal standards are indicated. The ceramide products and internal standards ASM-P, ASM-IS, GALC-P, GALC-IS, GBA-P, and GBA-IS undergo CID to give the common ammonium ion shown (m/z = 264). GLA-P, GLA-IS, GAA-P, and GAA-IS undergo neutral–loss CID to give four different secondary ammonium ions. Enzymatic reactions with DBS are shown by solid arrows, and CID is shown by dashed arrows. Reproduced from Ref. (7) with permission from the American Association for Clinical Chemistry (AACC).
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