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In-Depth Information
tumor tissue samples to correlate potential effi-
-
cacy parameters with EGFR, angiogenesis, and
downstream pathway activation for at least 17
total or phosphorylated cell signaling proteins,
including EGFR, pEGFR, pSTAT5, p38, p21,
PARP, Ki-67, and IL-8. Data will be correlated
with additional parameters including FcyR poly-
morphisms and tumor and serum cytokines.
analyzed by RPMA for correlations with protein
abundance. Six protein abundance ratios were
elevated after cognitive decline compared to the
stable, minimal cognitively impaired group:
BLVRB/BLVRA, estrogen receptor
/BLVRA,
HemeOxygenase1/BLVRA, MMP-9/BLVRA,
PDGFR Tyr716/BLVRA, and S100A7/BLVRA
(Mueller et al., 2010).
70
a
VALIDATION OF MASS
SPECTROMETRYeDISCOVERED
CANDIDATE BIOMARKERS BY
RPMA
CONCLUSIONS AND VISION FOR
THE FUTURE
fills a critical missing
component of molecular pro
RPMA technology
ling: quantitative
measurement of signal pathway proteins and
their
Validation of candidate biomarkers discovered
by mass spectrometry (MS) must use indepen-
dent sample sets that are separate from those
used for discovery. RPMA is particularly useful
if only a single validated antibody exists for the
biomarker. Based on this advantage, RPMA can
be used to validate mass spectrometry derived
biomarker candidate proteins.
70,71,100
For
adequately validated antibodies, RPMA has
a sensitivity in the picogram per mL with
clinical-grade precision.
76,97,100
Low molecular
weight serum protein fractions are likely to
contain proteins and peptides that have been
shed from the diseased tissue.
59,70
Serum-based
biomarkers are attractive as drug targets and for
screening because serum is often more readily
available and easier to collect compared to tissue.
Mueller et al. used liquid chromatography
e
MS/MS to identify candidate protein biomarkers
in LMW serum fractions from a community-
based cohort of patients with minimal cognitive
impairment before and after cognitive decline to
Alzheimer
ed forms.
RPMA is uniquely positioned for broad-scale
pathway activation mapping of clinical
material, as no other existing technology can
quantitatively measure the activation state
of so many drug targets at once with so little
material
d
thetypeofwhich .e,coreneedle
biopsies) underpin many predictive marker tri-
als.
3,5,8,9,14,16,17,34,41,48
e
50,52,60,61,75,80
e
82,86,98,101,102
Moreover, the compendium of FDA-cleared
targeted inhibitors is growing rapidly, with
the number of companion diagnostic markers
associated with each drug likewise expanding
quickly. In the near future, a physician will
have to know the drug target levels of perhaps
50 to 100 analytes, matched with the same
number of FDA-cleared drugs; thus RPMA is
uniquely positioned as a companion diagnostic
platform that can measure the activation state
of all of these at once. Measuring this class of
analytes, which contains the targets of molec-
ular inhibitors, provides functional information
about the disease state of cells that could not
be obtained by genetics or genomics. The need
to measure this class of protein analytes from
small biopsy samples will continue and expand
into the future (Mueller et al 2010
71
).
Every month, new disease-associated signal
pathway analytes are elucidated, new molecular
post-translationally modi
s disease.
70
Based upon their mass
spectrometry analysis, Biliverdin reductase B
(BLVRB) and S100A7, in addition to proteins in
the heme degradation and invasion pathway,
were selected for veri
'
cation by RPMA. Super
oxide dismutase, MMP-9, PDGFR Tyr716,
estrogen receptor
(ERA), biliverdin reductase
a
A (BLVRA)
and HemeOxygenase1 were
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