Development of a Multiplex Exoglycosidase Assay for Diagnosis of Oligosaccharidoses using Tandem Mass Spectrometry

Oligosaccharidoses are Lysosomal Storage Disorders (LSDs) that result from mutations in genes encoding exoglycosidases, leading to accumulation of unmetabolized N-linked oligosaccharides within lysosomes. Age at onset and rate of disease progression vary among patients. Diagnosis based solely on clinical presentation is often challenging because of overlapping clinical symptoms between these disorders. The aim of this research is to use tandem mass spectrometry (MS/MS) to establish a multiplex method to measure exoglycosidase activities, in dried blood spots (DBS), involved in the degradation of N-linked oligosaccharides using natural substrates. Current fluorometric assays for each exoglycosidase using specific 4-methylumbelliferyl (4MU) substrates allow enzyme activities to be determined separately from a variety of human tissue sample types. A universal buffer was established by comparing these assay conditions to allow multiplexing of the exoglycosidases in a single vial. Initial attempts to develop an enzyme activity assay using disaccharides as the starting substrate and by monitoring unique monosaccharide products by MS/MS after exposure to an enzyme source from cultured skin fibroblasts were unsuccessful due to interfering endogenous hexose isomers. Taking another approach, multiplexing was successfully demonstrated for beta-Galactosidase and beta-Hexosaminidase using alternative substrates. 4MU and paranitrophenol (PNP) conjugated to particular monosaccharides allowed 4MU and PNP products to be measured and enzyme activities to be calculated. Here, we provide a proof of principle that MS/MS technology can allow simultaneous multiplexing of several enzyme activities using distinctive starting substrates. A multiplex assay for the remaining exoglycosidases can still permit the development of an Oligosaccharidoses screening test to assist clinical diagnosis.