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.

In this study maltose, maltotriose and maltotetraose were for the first time, coupled to 4,4′-methylenedianiline (MDA). The aim of this preliminary work was to test the feasibility of oligo- and polysaccharide coupling to MDA and the characterization of the coupling products by high resolution mass spectrometry (MS). (+) nanoESI in combination with a quadrupole time of flight (QTOF) MS in full scan (MS) and MS/MS was optimized first on underivatized maltose, maltotriose and maltotetraose. The optimal screening and sequencing conditions were further applied to the MDA-functionalized oligosaccharides. The obtained results revealed a straightforward MS detection of the functionalized oligomers, high sequence coverage and a fragmentation pathway with the formation of B and Y ions as well as the complementary C and Z ions along with a typical cleavage of the aglycon. We consider that this methodology is fully applicable also to polydisperse mixtures of long chain polysaccharides, which due to the large number of components and their size require a systematic method of development and testing.

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