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

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2017-06, Vol.63 (6), p.1118-1126
• Main Authors: Liu, Yang, Yi, Fan, Kumar, Arun Babu, Kumar Chennamaneni, Naveen, Hong, Xinying, Scott, C Ronald, Gelb, Michael H, Turecek, Frantisek
• Format: Article
• Language: English
• Subjects: Assaying; Children; Chromatography; Chromatography, Liquid; Deuterium; Dried Blood Spot Testing - methods; Enzymatic activity; Enzymes; Humans; Infant, Newborn; Infants; Ions; Liquid chromatography; Lysosomal enzymes; Lysosomal storage diseases; Lysosomal Storage Diseases - blood; Lysosomal Storage Diseases - diagnosis; Lysosomal Storage Diseases - enzymology; Mass spectrometry; Mass spectroscopy; Medical screening; Methods; Mucopolysaccharidoses - blood; Mucopolysaccharidoses - metabolism; Mucopolysaccharidosis; Multiplexing; N-Acetylgalactosamine; N-Acetylgalactosamine-4-sulfatase; N-Acetylgalactosamine-6-sulfatase; Neonatal Screening - methods; Neonates; Neuronal ceroid lipofuscinosis; Neuronal Ceroid-Lipofuscinoses - blood; Neuronal Ceroid-Lipofuscinoses - diagnosis; Neuronal Ceroid-Lipofuscinoses - enzymology; Peptidase; Scientific imaging; Spectroscopy; Spots; Substrates; Tandem Mass Spectrometry
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2016.269167

We expanded the use of tandem mass spectrometry combined with liquid chromatography (LC-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). New substrates were prepared and characterized for tripeptidyl peptidase 1 (TPP1), α- -acetylglucosaminidase (NAGLU), and lysosomal β-glucuronidase (GUSB). These assays were combined with previously developed assays to provide a multiplex LC-MS/MS assay of 7 lysosomal storage diseases. Multiple reaction monitoring of ion dissociations for enzyme products and deuterium-labeled internal standards was used to quantify the enzyme activities. Deidentified DBS samples from 62 nonaffected 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), -acetylgalactosamine-6-sulfatase (GALNS), and -acetylgalactosamine-4-sulfatase (ARSB). The activities measured in the 7-plex format showed assay response-to-blank-activity ratios (analytical ranges) of 102-909 that clearly separated healthy infants from affected children. The new multiplex assay provides a robust comprehensive newborn screening assay for the mucopolysaccharidoses. The method has been expanded to include additional lysosomal storage diseases.