Environmental Engineering Reference
In-Depth Information
8.6 Chip-Based Mass Spectrometry for Rapid Diagnosis
of Schindler Disease
Schindler disease is a recently recognized lysosomal storage disorder caused by the
deficient activity of
-
N
-acetylgalactosaminidase (NAGA) enzyme. From the three
different phenotypes identified to date, the most severe form is the type I with an
infantile-onset neuroaxonal dystrophy. Schindler disease type II represents a milder
form with an adult-onset characterized by
angiokeratoma corporis diffusum
and
mild intellectual impairment, while the type III represents an intermediate form
with manifestations ranging from psychomotor retardation in infancy to a milder
autism and marked behavioral difficulties. The NAGA deficiency causes a
100 times higher concentration of
O
-glycans in urine than in healthy controls. For
this reason, screening, structural characterization and complete identification of
O
-
glycosylated amino acids and peptides extracted from patients
α
'
urine are of major
diagnostic importance.
Mass spectrometry based on nanofluidics have revealed in the last decade the
ability to discover in Schindler disease patient urine
O
-glycosylated amino acids
and
O
-glycopeptides as disease markers useful for a rapid diagnosis [
29
,
30
,
66
-
68
]. Besides, MS experiments based both on out-of-plane nanochip devices and
polymer thin microchips for screening and sequencing by CID of glycoconjugate
pools extracted and purified from the urine of Schindler disease patients and normal
controls were characterized by an unsurpassed sensitivity, throughput, reproduc-
ibility, quality and reliability of the data [
29
,
30
,
66
-
68
].
For mapping, sequencing and structural elucidation of a complex mixture of
O
-
glycosylated amino acids and peptides extracted and purified from the urine of a
healthy subject (denoted
Ty
), the thin chip polymer-based microsprayer coupled to
a hybrid QTOF MS has been used [
29
]. An aliquot of 10
μ
lof
O
-GalNAc
l
1
sample con-
centration was dispensed into the reservoir pasted over the inlet of the chip
microchannel as described in Sect.
8.2
.
The negative ion mode electrospray process was initiated at 2.8 kV ESI voltage
and 100 V potential of the sampling cone. The flow rate of about 200 nl min
1
together with the source parameters generated a constant and stable spray. The
spectrum combined over 20 scans (40 s), equivalent to a sample consumption of
0.66 pmol, exhibited a high signal/noise (S/N) ratio and yielded data on 26 different
saccharide components which were expressing the
O
-GalNAc-Ser/Thr core-motif
extended by either sialylation or fucosylation. The mixture was found to be
dominated by serine (Ser-) and threonine (Thr-) linked disialosaccharides with
chain lengths ranging from tetra- to octasaccharide.
In order to test the limit of microchip sensitivity for glycoconjugate detection,
Ty
solution was diluted in pure methanol yielding an aliquot at 1.25 pmol
glycosylated amino acids and peptides mixture having 5 pmol
μ
l
1
con-
μ
centration and further analyzed (Fig.
8.9
).
The signal was acquired for over 20 scans, which resulted in 0.16 pmol sample
consumption. Even under these limited concentration conditions, the potential of
