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molecular discovery and assessment of differen-
tially expressed proteins regulating H-Ras-
induced cardiomyopathy and can be applied to
serial tissue sections of similar or different origin.
To facilitate advanced molecular pro
the identi
ed proteins as potential biomarkers
can be further investigated in high-throughput
manner using multiplex quantitative immunoas-
says (i.e., ELISA, immuno-MRM) on larger
patient cohorts in clinical trials.
ling of
clinical specimens, we used the two-stage
organic/aqueous protocol to develop an individ-
ualized approach for cancer biomarker
discovery using RCC as a model disease. 3 Tissue
specimens were homogenized using mechanical
grinder followed by ultrasonication. This proteo-
mic approach relies on tissue-directed subtractive
proteomics to detect tumor proteins in peripheral
blood that may be utilized as potential markers
for diagnosis and targeted treatment in a patient
diagnosed with RCC. Shotgun proteomics was
used to analyze tumor, normal adjacent tissue,
and blood obtained from a single patient diag-
nosed with RCC. The analysis resulted in a total
of 1,275 proteins identi
CONCLUDING REMARKS
Sample preparation is still a major issue in
large-scale molecular pro
ling of clinical tissue
specimens. The tissue sample preparation work-
flow is complex and often encompasses a greater
number of experimental steps and sample vari-
ables when compared to processing of cultured
cells. For biomarker discovery, it is critical that
the protein complement of diseased and healthy
tissue is pro
led directly in vivo , at the site of
pathological process. It is more likely that molec-
ular pro
les/phenotypes obtained from tissue
specimens may lead to distinct insights that are
not readily evident when analyzing in vitro
cultured cells and may signi
ed in tumor and 1,281 in
normal adjacent tissue, of which 1,073 proteins
were commonly found in both tissues. Subtrac-
tive proteomics revealed a total of 202 proteins
detected only in tumor. Of these, eight proteins
were identi
cantly accelerate
the discovery of disease biomarkers. Also, the
likelihood of discovering low-abundance
markers using exclusively MS-based pro
ed in the plasma by meeting the
following criteria: (1) identi
ling
of peripheral blood is low due to the volume
dilution effect. The type of tissue, the choice of
homogenization technique, and the extraction
buffer are critical for experimental design of
any tissue-based proteomic study. Prospectively
collected FF tissue specimens are currently the
gold standard for MS-based proteomic pro
ed in tumor but
not in normal tissue, (2) identi
ed in tumor
and matching plasma, and (3) possessing higher
spectral count in tumor than plasma. Three of
the eight identi
ed proteins (i.e., cadherin-11,
pyruvate kinase, and vascular cell-adhesion
molecule-1) were previously shown to be impli-
cated in RCC tumorogenesis. Four of the identi-
ling.
There are no
firm criteria for selection of tissue
homogenization technique; it should be done
on a case-by-case basis driven primarily by phys-
ical characteristics of the tissue under study. It is
often necessary to combine two methods for
optimal results (e.g., mechanical grinding and
sonication. 3,44 Importantly, the choice of extrac-
tion buffer may affect the proteome coverage
more than the choice of the tissue homogeniza-
tion method. 45 Innovative approaches relying
on subtractive proteomics and concomitant
fied proteins (i.e., cadherin-5, cadherin-11,
DEAD-box protein-23, and pyruvate kinase)
were cross-validated by Western blot analysis
con
rming their presence in the blood of the
patient under the study, as well as in the blood
of four other patients diagnosed with RCC.
These results provide the
first evidence that in-
depth tissue-directed subtractive proteomics is
capable of identifying tumor-derived proteins
in peripheral blood of a patient diagnosed with
cancer. Importantly, the general applicability of
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