Testing the feasibility of fully automated chip‐based nanoelectrospray ionization mass spectrometry as a novel tool for rapid diagnosis of Fabry disease

Abstract

Fabry condition, a lysosomal storage disease (LSD) is characterized by the absence or reduction of the α-galactosidase A activity. Recently, a new diagnostic method for detection of α-galactosidase activity from dried blood spots (DBS) using a chemical substrate and quantification of reaction mixture was developed. To improve this method in the terms of automation, reproducibility, sensitivity, and data reliability, we introduce here an innovative analytical approach based on chip-nanoESI MS. The α-galactosidase assay products derived from DBS of 11 healthy donors and 11 Fabry disease patients were analyzed by NanoMate robot coupled to a high-capacity ion trap MS. Confirmation and structural analysis of the reaction products was achieved by CID and electron transfer dissociation (ETD) MS/MS. The cleavage of a substrate GLA-S generated a product, GLA-P, which was quantified related to an internal standard GLA-IS. Comparative patient versus control analysis indicated a 13-fold reduction in GLA-P/GLA-IS ratio in the case of the patients. Moreover, our method provided direct data on the enzyme, from which it was for the first time possible to discriminate between the patients lacking the enzyme and those presenting a less active one. GLA-IS and GLA-P were confirmed by CID/ETD, which applied together, increased considerably the sequence coverage and provided complementary information for unambiguous product identification. The present chip-nanoESI CID and ETD MS(n) strategy introduced here for first time in LSD diagnosis, provided a maximum confidence in assay product identification, a high sensitivity, speed of analysis, and result reproducibility.

Nanofluidics-Based Mass Spectrometry. Applications for Biomarker Discovery in Lysosomal Storage Diseases

“…In order to avoid the generation of false-negative or false-positive response, it is necessary to ensure through the implemented methods a high level of reproducibility and sensibility necessary for accurate comparative screening patient vs. healthy control, as well as to avoid the carry-over from sample to sample which might occur during successive ESI infusions. In 2013, a novel platform for rapid and reliable diagnostic of Fabry disease, that eliminates all the drawbacks of the DBS-MS method mentioned above, have been successfully implemented [63] and it is based on enzyme assay and fully automated chip-nanoESI MS, collisioninduced dissociation (CID) and electron transfer dissociation (ETD) MS/MS for a total of 13 DBSs, 11 from healthy donors and 2 from Fabry patients. The enzymatic assay workflow comprised five stages: (1) obtaining the DBSs by uniform spotting 75 μL of whole blood onto Whatman 903 ® specimen collection paper, drying at room temperature and cutting with a 3 mm diameter puncher from the middle of the circle, then storing them at 4 C; (2) enzymatic extraction from DBS using an aqueous buffer solution; (3) enzymatic reaction between the DBS α-galactosidase extracts and the substrate/internal standard (GLA-S/GLA-IS) cocktail -molar ratio 500:1 -resulting in cleavage off the terminal galactose from GLA-S generating the product (GLA-P).…”

“…In the last 5-6 years, different MS-based strategies for monitoring the α-galactosidase A (GLA) enzyme in dried blood spots (DBS) [58,[61][62][63], as well as for measurement of the storage products in plasma [64] or urine have been developed and implemented. Despite the facts that were capable to provide early detection of LSDs in general and of Fabry disease in particular, false-negative results [65] have been reported in a number of previous studies based on DBS assays.…”

Dried blood spots: Concepts, present status, and future perspectives in bioanalysis

“…In diagnosis of hypovitaminosis D LC-MS/MS [221] 85. α1-antitrypsin genotypes Early diagnosis of hereditary disorder, associated with early risk of onset chronic obstructive pulmonary disease and liver dysfunction Fluorometric elastase inhibition assay [222] 86. α-galactosidase Dignosis of Fabry disease Chip-based nanoelectrospray ionization mass spectrometry [223] 87. α-iduronidase Mucopolysaccharidosis I diagnosis Enzyme assay [224] A. Sharma et al samples as estimation of several supplementary validation parameters like analyte stability on cards during drying and storage, the effect of hematocrit, spot homogeneity, and analyte elution efficiency from the card are also mandatory.…”

Advances in Mass Spectrometry for Glycoscreening and Sequencing in Biomedical Research

“…In recent years, several MS-based strategies for monitoring the α-galactosidase A enzyme in Dried Blood Spots (DBS) [37,38], as well as for measurement of the storage products in plasma or urine have been developed. In 2013, a novel platform for rapid and reliable diagnostic of Fabry disease, based on enzyme assay and fully automated chip-nanoESI MS, CID and ETD MS/MS was reported [38].…”