Tandem Mass Spectrometry Has a Larger Analytical Range than Fluorescence Assays of Lysosomal Enzymes: Application to Newborn Screening and Diagnosis of Mucopolysaccharidoses Types II, IVA, and VI

Abstract

Background: There is interest in newborn screening and diagnosis of lysosomal storage diseases because of the development of treatment options that improve clinical outcome. Assays of lysosomal enzymes with high analytical range (ratio of assay response from the enzymatic reaction divided by the assay response due to nonenzymatic processes) are desirable because they are predicted to lead to a lower rate of false positives in population screening and to more accurate diagnoses.

Methods: We designed new tandem mass spectrometry (MS/MS) assays that give the largest analytical ranges reported to date for the use of dried blood spots (DBS) for detection of mucopolysaccharidoses type II (MPS-II), MPS-IVA, and MPS-VI. For comparison, we carried out fluorometric assays of 6 lysosomal enzymes using 4-methylumbelliferyl (4MU)-substrate conjugates.

Results: The MS/MS assays for MPS-II, -IVA, and -VI displayed analytical ranges that are 1-2 orders of magnitude higher than those for the corresponding fluorometric assays. The relatively small analytical ranges of the 4MU assays are due to the intrinsic fluorescence of the 4MU substrates, which cause high background in the assay response.

Conclusions: These highly reproducible MS/MS assays for MPS-II, -IVA, and -VI can support multiplex newborn screening of these lysosomal storage diseases. MS/MS assays of lysosomal enzymes outperform 4MU fluorometric assays in terms of analytical range. Ongoing pilot studies will allow us to gauge the impact of the increased analytical range on newborn screening performance.

Fig. 1. Structures of the substrates for assaying GALNS, ARSB, and I2S

The proton shown hydrogen bonded to the 2 carbonyl groups is the proposed site of gas phase protonation in the electrospray ion source. Internal standards lack the sulfate and contain 5 deuteriums on the benzoyl group.

Fig. 2. Enzymatic reactions for I2S, GALNS, and ARSB

Coupling enzymes to convert the direct product of the lysosomal enzyme to the free aglycone are shown (this step is omitted for some variations of the assays described in the main text). The use of (Z)-Pugnac to block the action of human hexosaminidase A on the GALNS and ARSB substrates is also shown.

Fig. 3. Enzyme activities in random newborns and LSD-affected patients measured using LC-MS/MS

Note the difference in y-axis scales. Mean activity is shown as a horizontal line.