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assay applicability for low-abundance biomarker
veri
combination with further fractionation also has
great impact on multiplexing capabilities and
sample throughput.
To deal with the sample complexity issue and
improve sensitivity for plasma samples, a better
strategy would include targeted sample prepara-
tion in which low-abundance targets can be
enriched and the sample matrix can be simulta-
neously simpli
cation studies in plasma with emphasis
being placed on minimum impact on assay repro-
ducibility and throughput while improving assay
sensitivity.
SAMPLE ENRICHMENT
STRATEGIES FOR IMPROVING
B
IOMARKER VERIFICATIO
N
ed. Targeted sample preparation
strategies can either enrich for target proteins or
enrich for peptides that act as surrogates for
protein quantitation. Common enrichment strat-
egies performed at the protein level include the
use of antibodies, gel electrophoresis, or off-gel
fractionation. Unfortunately, these techniques
are generally time consuming and dif
To improve detection sensitivity in plasma
and serum samples, the
first sample preparation
strategy that generally comes to mind is immu-
nodepletion of highly abundant proteins, a tech-
nique that has been widely applied to biomarker
discovery studies.
24,25
The reproducibility and
the ef
cult to
multiplex with low sample throughput. A recent
approach termed stable isotope standard
capture with antipeptide antibodies (SISCAPA)
has shown great potential for low-abundance
biomarker veri
ciency of immunodepletion strategies for
depletion of the intended abundant proteins
have been thoroughly investigated and demon-
strated.
24
e
26
Thus the depletion strategy in
combination with further fractionation using
techniques such as strong cation exchange chro-
matography have been applied to plasma
samples prior to their analysis by MRM-MS.
This combination strategy has successfully
achieved ng/ml LOQs for clinically relevant
biomarkers, including PSA and cardiovascular
biomarkers in patient samples.
27
e
29
Although
immunodepletion columns can remove the tar-
geted abundant plasma proteins ef
cation when applied to enrich
for targeted peptides. The peptides of interest
are enriched from digested plasma samples
that are spiked with known amounts of their
stable isotope-labeled internal standard counter-
parts. The enrichment uses immobilized anti-
bodies generated against speci
c peptides. The
peptides are released from the antibody and
then quanti
ed using MRM-MS.
31
White-
aker et al.
32
presented an improved SISCAPA
approach through automating the process using
a magnetic-bead-based platform capable of tar-
geting nine peptides in the same assay. The posi-
tive or negative impact of the multiplexing
format on peptide target recovery seems to
depend on the individual peptide, but the over-
all performance of the 9-plex panel was not
adversely affected. While using a 10
ciently and
reproducibly, there are caveats that need to be
considered when using this sample preparation
strategy. In a recent study by Tu et al., only 23
proteins at less than ng/ml level were identi
ed
after immunodepletion was performed.
26
In
addition, the depleted high-abundance proteins
could potentially carry clinically relevant
biomarkers either due to direct interaction or
due to the so-called sponge effect. Gundry
et al. reported that 9 of the 26 albumin-
associated proteins that are considered potential
biomarkers were identi
l plasma
sample, the multiplexed SISCAPA process
provided enrichment of 100- to 1,000-fold with
overall quantitation median CVs of 12.6%. This
automated and multiplexed sample enrichment
approach allows for quanti
m
ed only in the albumin-
enriched fraction.
30
The nontargeted sample
preparation strategy of
cations of proteins
in plasma at ng/ml level without any deple-
tion and fractionation steps. Additionally, as an
immunodepletion in
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