Environmental Engineering Reference
In-Depth Information
intensity. The higher abundance of the disialylated saccharides toward the
monosialylated species demonstrated the feasibility of the optimized chipESI
FTICR MS approach to exhibit only a very low degree of in-source fragmentation
related to the labile sialic acid moiety, and therefore to satisfy the fundamental
criteria for detection of intact sialylated glycoconjugates.
For exploring the limits of sensitivity at which the level of mixture complexity
could still be reliably assessed, a broad study on four successive dilutions from
20 up to 3 pmol
l
1
(calculated using the average relative molecular mass of the
components,
M
r
¼
2,500) was conducted by chipESI FTICR MS method optimized
in negative ion mode for the
Gy2
mixture of glycopeptides. In comparison with the
chipESI FTICR spectrum of the normal urine glycopeptides, in the spectrum of the
patient urine a higher content of pentasaccharides was observed, while
hexasaccharides linked to Ser and Thr bearing two sialic acid moieties were
detected at higher abundance. Moreover, a nonasaccharide bearing three sialic
acid moieties assigned to the sodiated dehydrated Neu5Ac
3
Hex
2
HexNAc
4
-Ser
was for the first time detected with a mass accuracy of 4.6 ppm.
The potential of the chipESI FTICR MS method to exhibit not only a high
sensitivity, high mass accuracy and resolution but also superior reproducibility of
the experiments was also demonstrated in this study; no significant difference with
respect to the ionic species observed between the spectra resulted from 12 and
6 pmol
μ
l
1
sample concentration (Figs.
8.10a, b
) was observed.
Unlike conventional capillary-based nanospray characterized by poorer repro-
ducibility of the glass-capillaries shape and signal interruption due to blockages of
the tip, polymer chip produces a spray of high stability over a long time frame of
signal acquisition, even for complex biological analytes. This aspect is more clearly
illustrated by the spectrum in Fig.
8.10b
, where 100 scans were acquired for a
sample concentration of only 6 pmol
μ
l
1
.
The coupling of fully automated chip-based nanoESI system (NaoMate robot) to
high-performance mass spectrometry was introduced in clinical analyses and in
particular for LSDs, almost 10 years ago [
66
-
68
].
In 2004, the newly conceived NanoMate/FTICR coupling was tested for high-
performance mapping and sequencing by sustained off-resonance collision-induced
tandem mass spectrometry (SORI-CID MS
2
)of
O
-glycosylated sialylated peptides
and amino acids from the urine of a patient suffering from Schindler disease and from
the urine of a healthy age-matched individual for comparative assay [
66
]. The
mixtures were dissolved in methanol to different concentrations and transferred to
the glass plate of the NanoMate
μ
100 robot. The initiation of the electrospray
required a starting back pressure of about 6,200 Pa, which, in order to enhance the
stability of the spray, was slightly decreased to lower values during acquisition
[
66
]. For the initiation of the chip spray and the efficient transferring of the ionic
species into the mass spectrometer, a fine adjustment of the NanoMate chip position
with respect to the FTICR ion transfer capillary has been reported to be a critical step.
An extensive study on the limit of sensitivity of the method was carried out by
stepwise dilutions to 5 pmol
™
l
1
l
1
, 1 pmol
l
1
μ
(Fig.
8.11
), 2.5 pmol
μ
μ
and
l
1
, respectively; the highest sensitivity obtained in this set of experiments
0.5 pmol
μ
