Environmental Engineering Reference
In-Depth Information
[M+H
+
]
+
GLA-S
Intens.
Intens.
6
x10
6
x10
646.0
6
6
Globotriaosylceramide
5
5
GalNAc(
ʲ
1
ₒ
3)
Gal(
ʱ
1
ₒ
4) Gal(
ʲ
1
ₒ
4)Glc(
ʲ
1
ₒ
1')Cer
4
4
3
3
-galactosidase
ʱ
[M+Na
+
]
+
[M+H
+
]
+
GLA-S
2
2
GLA-P
484.1
668.0
[M+K
+
]
+
[M+H
+
]
+
GLA-S
GLA-IS
684.1
1
1
489.1
719.0
535.8
987.9
590.0
0
0
600
800
1000
1200
1400
m/z
Fig. 8.8 Fully automated chip-nanoESI on a NanoMate robot coupled to HCT MS of the assay
products after enzymatic reaction in a DBS sample from a patient diagnosed with Fabry disease;
in vivo, the major non-degraded substrate is globotriaosylceramide. Experiment conditions as in
Fig.
8.7
. Reprinted with permission from [
63
]
The low intensity signal of GLA-P in Fig.
8.8
illustrates a diminished activity of
the enzyme, while the lack of protein signals indicates the presence of the enzyme
in a quantity which is below even the detection limit of the chip-nanoESI HCT
MS. There were differences also regarding the GLA-P/GLA-IS ratio, which in
Fig.
8.8
was only 2. This 13-fold reduction in GLA-P/GLA-IS ratio calculated
according to absolute intensity of the signals from MS screening in the comparative
control vs. patient highlighted that the platform can reliably monitor the activity/
expression of
α
-galactosidase in Fabry patients by chip-based nanoESI
MS. Moreover, confirmation of GLA-P as well as the GLA-IS structures was
further carried out by extensive structural characterization using two complemen-
tary fragmentation techniques, CID and ETD, the latter being for the first employed
in rapid diagnosis of LSDs.
The sensitivity and reproducibility of the NanoMate HCT MS platform enable
discrimination between different Fabry cases, an aspect with major clinical impor-
tance when choosing an appropriate therapy. For instance, this rapid detection of
rare diseases tool exhibit the capability to distinguish the situations in which the
GLA enzyme is completely absent from those in which the enzyme is present at the
trace level, as well as between the patients unable to produce the GLA enzyme from
those that produce an inactive GLA enzyme.
