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enrichment strategy, the SISCAPA approach can
be easily applied to larger sample volumes to
further improve detection sensitivity. As demon-
strated in the same study, applying the method
to 1 ml of plasma resulted in limits of detection
(LODs) and LOQs that were further decreased
to pg/ml protein level for most targets while
still maintaining assay performance. A potential
drawback for the SISCAPA process is that not
all peptides are antigenic, as only 9 out of the
original 15 peptides used in the study generated
working antibodies. Multiple peptides per
protein could potentially assist in generating
useful antibodies, although the cost and overall
lead time will also increase. To evaluate the
feasibility of developing large-scale biomarker
quantitation assays based on SISCAPA in combi-
nation with MRM-MS, Whiteaker et al. success-
fully generated antibodies for such working
assays with sensitivity in low ng/ml range
(enrichment from 10
been widely used for various sample types
including human sera and urine. 35 Although it
has not been exploited in combination with
MRM-MS for plasma and serum samples, it
has recently been applied in combination with
an MRM-MS assay for quantitation of low-
abundance proteins in patient ovarian cancer
ascites digests. 36 A multiplexed MRM-MS assay
of
five proteins including kallikrein 6 (in ng/ml
level) were developed and the quantitation
values obtained for this protein after combina-
tory peptide library treatment correlated
well with those from earlier ELISA results
(R 2
0.988). However,
the
reproducibility,
ΒΌ
ef
ciency, and overall applicability of this
sample enrichment strategy for multiple targets
in a highly complex matrix such as plasma
obviously must be further investigated and vali-
dated before it can be applied for protein
biomarker veri
cation purposes.
l plasma) using up to 5
peptides per protein for 89 cancer relevant
proteins in a year with 100% per-protein and
54% per-peptide success rates. 33 The multiplexed
SISCAPA MRM-MS approach was also evalu-
ated in another study for its interlaboratory
performance to quantify proteins in plasma at
or below the 1 ng/ml level and demonstrated
acceptable
m
c sample preparation
enrichment, many strategies have been devel-
oped to target speci
As
for PTM-speci
cations of interest
during the proteomic discovery phase. Technol-
ogies have only just advanced enough for deeper
investigation into the functional signi
c modi
cance of
the PTMs in potential biomarker discovery. 17 e 19
More investigations into enrichment strategies
targeting PTMs in combination with MRM-MS
will probably be evaluated and performed once
additional peptides with PTMs are identi
reproducibility
for
veri
cation
purposes. 34
Alternatively, another af
nity enrichment
strategy based on combinatorial hexapeptide
ligand library coupled to beads shows
the potential to achieve enrichment of low-
abundance proteins while minimizing sample
dynamic range in a nontargeted manner
compared to SISCAPA. 35 The nontargeted
manner is in the sense that it is not targeted at
any speci
ed as
potential biomarkers.
MASS SPECTROMETRY
BASED
STRATEGIES TO IMPROVE
B IOMARKER VERIFICATIO N
e
c protein or proteins but binds
proteins up to its capacity. The combinatorial
peptide ligand library reduces sample dynamic
range by equalizing the concentrations of all
proteins by capturing them up to its binding
capacity for all proteins, resulting in the overall
enrichment of low-abundance proteins. It has
In addition to all the sample preparation
efforts being developed to improve the sensi-
tivity of targeted MRM-MS approaches, Fortin
et al. 37 reported an MS-based strategy (termed
MRM 3 ) to improve limit of quantitation by
taking advantage of the ability of a hybrid triple
quadrupole/linear ion trap mass spectrometer
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