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matrices, must be carefully evaluated. If success-
ful, it would greatly improve both the speed and
the accuracy of future large-scale biomarker veri-
the reliability of the biomarkers veri
ed. What
'
s
more,
city of the bio-
markers for the targeted condition was also eval-
uated and further veri
the differential speci
fication efforts using the targeted MRM-MS
approach.
ed using plasma samples
from subjects with confounding conditions
instead of only healthy controls. It is also clear
from these two studies that the combination of
immune-depletion and strong cation exchange
fractionation is unavoidable when dealing with
plasma samples for real biomarker veri
SELECTED BIOMARKER
VERIFICATION APPLICATIONS
BASED ON MRM-MS
cation
purposes even when using the more sensitive
and speci
Since the establishment of MRM-MS as a
promising strategy for reproducible and accu-
rate high throughput biomarker quantitation, it
has been applied in several recent studies to
facilitate large-scale integrated LC-MS-based
investigation of biomarkers from discovery stage
to veri
c SID-MRM-MS approach, which
certainly has an impact on both sample through-
put and results precision. The study by
Whiteaker et al. also developed SISCAPA
MRM-MS assays for 35 very-low-abundance
biomarker candidates with about 85% assay
development success rate and excellent assay
working performance. The above-mentioned
experimental details are the issues to be dealt
with in any biomarker veri
cation stage.
3
e
8,56
Studies by Addona
et al.
5
and Whiteaker et al.
6
are two excellent
examples of work carefully designed and per-
formed in an integrated
flow from identify-
ing potential biomarkers to re
ow.
These two studies not only demonstrated the
capability and applicability of the current mass
spectrometry-based approaches for dealing
with the large numbers of potential biomarkers
in such complex and promising matrix as
plasma, but also provided blueprints for future
advancement in biomarker veri
cation work
ned credible
biomarker veri
cations. The study by Addona
et al. identi
ed 121 potential biomarkers through
the traditional biomarker discovery route where
in-depth proteomics analysis and label-free
quantitation were performed on three patient
samples through extensive fractionation. The
study by Whiteaker et al., on the other hand,
integrated results from 13 earlier independent
genomic and proteomic data sets and identi
cation work-
flow in the proteomics community. Besides
working with peripheral plasma, a recent study
veri
ed
1,908 potential biomarkers. Both studies used
strategies to qualify and re
ed potential male infertility biomarkers in
seminal plasma using SID MRM-MS approach.
7
It basically followed a similar work
ne many potential
biomarker candidates (121 and 1,908, respec-
tively) to ones demonstrating reliable differential
potential in peripheral plasma (52 and 91,
respectively) before further verifying them using
SID MRM-MS. Both studies used pooled plasma
for the biomarker quali
ow as the
previously described work but without any
sample fractionation; 79 potential differentiation
biomarkers were identi
ed using spectral count
information, further quali
ed in seminal plasma
samples using relative MRM-MS quantitation
normalized with one internal standard down to
18 targets to be veri
cation stage to focus on
biomarkers typical of the condition investigated
but individual plasma samples at the
ed by absolute quantitation
based on SID MRM-MS. A biomarker panel of
16, 3, and 11 azoospermia biomarkers out of
the 18 veri
-
cation stage to verify their applicability at indi-
vidual level as the condition markers. Different
samples were used at different stages of the
veri
final verifi-
ed using 30 seminal plasma samples
was proposed to be validated further in larger
cohorts in the future to differentiate normal,
cation work
ow, which further improves
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