Multiplex Tandem Mass Spectrometry Enzymatic Activity Assay for Newborn Screening of the Mucopolysaccharidoses and Type 2 Neuronal Ceroid Lipofuscinosis

Abstract

Background We have expanded the use of tandem mass spectrometry combined with liquid chromatography (HPLC-MS/MS) for multiplex newborn screening of seven lysosomal enzymes in dried blood spots (DBS). The new assays are for enzymes responsible for the mucopolysaccharidoses MPS-I, -II, -IIIB, -IVA, -VI, and −VII) and type 2 neuronal ceroid lipofuscinosis (LINCL). Methods New substrates were prepared and characterized for tripeptidyl peptidase 1 (TPP1), α-N-acetylglucosaminidase (NAGLU), and lysosomal β-glucuronidase (GUSB). These assays were combined with previously developed assays to provide a multiplex HPLC-MS/MS assay of seven lysosomal storage diseases (LSDs). Multiple reaction monitoring (MRM) of ion dissociations for enzyme products and deuterium-labeled internal standards was used to quantify the enzyme activities. Results Deidentified DBS samples from 62 non-affected newborns were analyzed to simultaneously determine (run time 2 min per DBS) the activities of TPP1, NAGLU, and GUSB, along with those for α-iduronidase (IDUA) iduronate-2-sulfatase (I2S), N-acetylgalactosamine-6-sulfatase (GALNS), and N-acetylgalactosamine-4-sulfatase (ARSB). The activities measured in the 7-plex format showed analytical ranges of 102-909 that clearly separated healthy infants from affected children. Conclusions The new multiplex assay provides a robust comprehensive newborn screening assay for the mucopolysaccharidoses, which is the most rapid method reported to date. The method is shown to be expandable to include additional LSDs including neuronal ceroid lipofuscinosis. The assay is also useful for biochemical diagnosis and prognosis studies.

Validation of Liquid Chromatography-Tandem Mass Spectrometry-Based 5-Plex Assay for Mucopolysaccharidoses

“…In this study, we established an enzyme activity assay using LC-MS/MS for simultaneous pretreatment and measurement of five types of enzymes lacking in MPS I, MPS II, MPS IIIB, MPS IVA, and MPS VI. This work is an extension of a previous study by Liu et al [30]. In the present study, we developed the analytical method of better LC separation by changing the analytical time from 2 min to 6 min to improve measurement sensitivity of enzyme activities for five MPSs including MPS I and II.…”

“…The IS and substrate of each MPS were obtained from PerkinElmer (Waltham, MA, USA; Figure 3 and Table 3). Substrate concentrations were determined as described previously [30]. LC-MS grade methanol, acetonitrile, and ultrapure water were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).…”

“…Therefore, column separation is necessary to analyze enzyme activities for these MPS types. Liu et al reported a multiplex liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of the enzyme activities of MPS I, II, IIIB, IVA, VI, and VII [30]. However, the report did not include the results of analysis with actual samples for MPS I.…”

Contribution of tandem mass spectrometry to the diagnosis of lysosomal storage disorders

“…A kit for NBS of FD, GD, KD, NPAB, PD, and MPS I is commercialized by PerkinElmer (Indianapolis, IN, USA). Extension of these assays is described for MPS II, IIIB, IVA, VI, and VII, and CLN2 (Liu et al 2017).…”

The role of tandem mass spectrometry in the diagnosis of inherited metabolic diseases

“…Тест является высокоспецифичным для каждого конкретного заболевания, поскольку применяются субстраты, близкие по своему строению к натуральным. Проходит апробацию новая тест-система для выявления других ЛБН (мукополисахаридозов типов II, III, VI, IVA, VII, нейронального цероидного липофусциноза 2-го типа) и в ближайшие годы ожидается ее появление [32], а в перспективе следует ожидать создания тест-систем для диагностики и многих других ЛБН.…”