High-Throughput Assay of 9 Lysosomal Enzymes for Newborn Screening

Abstract

BACKGROUND There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the availability of treatments. Pilot studies have used tandem mass spectrometry with flow injection of samples to achieve multiplex detection of enzyme products. We report a multiplexing method of 9 enzymatic assays that uses HPLC-tandem mass spectrometry (MS/MS). METHODS The assay of 9 enzymes was carried out in 1 or 2 buffers with a cassette of substrates and internal standards and 1 or 2 punches of a dried blood spot (DBS) from a newborn screening card as the source of enzymes. The pre–HPLC-MS/MS sample preparation required only 4 liquid transfers before injection into a dual-column HPLC equipped with switching valves to direct the flow to separation and column equilibration. Product-specific and internal standard–specific ion fragmentations were used for MS/MS quantification in the selected reaction monitoring mode. RESULTS Analysis of blood spots from 58 random newborns and lysosomal storage disease–affected patients showed that the assay readily distinguished affected from nonaffected individuals. The time per 9-plex analysis (1.8 min) was sufficiently short to be compatible with the workflow of newborn screening laboratories. CONCLUSIONS HPLC-MS/MS provides a viable alternative to flow-injection MS/MS for the quantification of lysosomal enzyme activities. It is possible to assay 9 lysosomal enzymes using 1 or 2 reaction buffers, thus minimizing the number of separate incubations necessary.

Newborn screening for lysosomal storage disorders

“…25,26 The switch to MS/MS was possible because, in collaboration with Chamoles, Michael Gelb, C. Ronald Scott, and others developed enzyme-specific substrates, synthesized isotopically labeled substrates, and measured the products of various enzyme reactions simultaneously in DBS with MS/ MS. 27 Since the first description of their Flow Injection Analysis (FIA) MS/MS assay, modifications including the number of enzymes, sample preparation steps, and the addition of liquid chromatography (LC) have been reported and applied in both limited studies and routine NBS. [28][29][30][31][32][33][34][35][36][37][38][39] Another analytical multiplex approach to NBS for LSDs, also based on Chamoles' fluorometric enzyme activity assays, was developed by Advanced Liquid Logic, Inc. (ALL; now called Baebies Inc.). 40 Their procedure has the ability to simultaneously perform up to 5 different enzyme assays on a single DBS.…”

Profile of alglucosidase alfa in the treatment of Pompe disease: safety, efficacy, and patient acceptability

“…19 High-throughput mass spectrometry methods are used for the implementation of newborn screening for lysosomal storage disorders (including Pompe disease) in pilot studies in Taiwan, North America, and Europe. [20][21][22][23] Currently, the "gold standard" diagnostic for Pompe disease is the GAA assay performed on skin fibroblasts or muscle biopsy followed by DNA analysis 24,25 with detection of homozygote or compound heterozygote mutations in the GAA gene. To date more than 300 different mutations have been found, 26 without strict genotype-phenotype correlation.…”

Mass spectrometry in clinical chemistry: the case of newborn screening

“…Some methods were developed for the detection of lysosomal storage disorders (LSDs) and even if for some of them a reliable and relatively simple test is available, a reasonable associated therapy could be still not available [29][30][31][32][33][34][35][36][37][38]. It is not the task of this writer to produce a statement that the LSDs should be included in newborn screening panels, but some of these disorders seem to meet the criteria for inclusion, including basal availability of a simple test, a combination therapy that modifies G. la Marca / Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx-xxx the natural course of the disease, early diagnosis that allows genetic counseling and prenatal diagnosis in families with an affected baby.…”