Biology Reference
In-Depth Information
SISCAPA
stable isotope standard capture with
antipeptide antibodies
QconCAT
facilitate the most thorough and unbiased sample
analysis. Therefore, only a limited number of
clinical samples of various sample types including
body
quanti
cation concatamer
PSAQ
protein standard absolute quanti
cation
uids and tissues are analyzed inmost cases,
due to the low-throughput nature of the discovery
phase. The potential biomarker candidates identi-
INTRODUCTION
fied at the discovery stage have a high false
positive rate due to the limited number of samples
analyzedandanalytical variation causedby exten-
sive sample preparation. Thus, before these
biomarkers can be developed into useful clinical
assays, they have to be validated as a true
biomarker for the intended condition and their
sensitivity and speci
Biomarker discovery has always been of great
interest to the clinical research community,
owing to the value of biomarkers in both diag-
nostics and therapeutic monitoring. In the year
2011 alone, there were over 44,000 publications
related to the term
in PubMed.
Arguably, the protein biomarker discovery
process has been greatly improved due to tech-
nology development, especially with the
advancement of mass spectrometers in the last
decade, leading to the exponential increase in
potential biomarkers presented in the scienti
“
biomarker
”
city must be established.
Biomarker validation requires a very large
number of clinical samples (e.g.,
1,000) com-
>
100).
2
Bio-
marker validation requires a higher threshold
of certainty than either discovery or veri
pared to the discovery stage (e.g.,
<
ca-
c
literature. As noted in a recent review by Ander-
son, even with great interest and plenty of fund-
ing, only an average of about 1 protein per year in
recent years eventually ends up as part of a well-
characterized assay utilized by the biomedical
community.
1
The paucity of validated bio-
markers is due to lack of an established accurate,
reproducible, and high-throughput protein quan-
titative assay with multiplexing capability. There-
fore, veri
tion.
It
is important
to establish sensitivity
and speci
city of the biomarker; its levels
must be measured against many thousands of
samples in which its variation within the tar-
geted population is precisely re
ected. This
cohort must include controls not only of
healthy individuals but also of those with
phenotypically similar conditions but with
a different underlying mechanism to that being
studied. Before all that effort, assays have to
be developed to bridge the gap between the
many potential biomarker candidates identi
nement of these potential
biomarkers into some credible few that are in
fact speci
cation and re
ed
during discovery phase and the validation
stage. Between the discovery and validation
phases comes veri
c indicators of any biological state of
interest to be validated and developed into
reliable clinic assays (usually antibody-based
assays) has become the new bottleneck for these
newly discovered potential protein biomarkers
and their application in medicine.
Mass spectrometry (MS) has played an instru-
mental role in the biomarker discovery stage.
Most potential biomarkers reported in the lite-
rature over the last decade are discovered
through extensive sample fractionation followed
by MS analysis. For MS-based protein biomarker
discovery, extensive sample procedure and
data-dependent acquisition are generally used to
cation.
2
For biomarker veri
cation, the data acqui-
sition strategy targets speci
c potential bio-
markers instead of measuring as many species
as possible. The goal of the veri
cation stage
is to select those potential biomarkers that
have the necessary speci
city and sensitivity
to potentially pass a
final validation phase.
This phase requires analyzing several hundred
samples but, unlike the discovery phase, it is
focused on a smaller number of prede
ned ana-
lytes (i.e.,
tens) and measures the absolute
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