Biology Reference
In-Depth Information
PROTEOME PROFILING WITH
PAcIFIC
approximately 10% of women will suffer from
the disease at some point in their lifetime.
28
Improvements in breast cancer treatment over
the last few decades have led to an overall
decrease in mortality of the disease, with adju-
vant anti-estrogen therapies (e.g., tamoxifen)
effectively treating a majority of estrogen
receptor (ER)
e
positive breast cancer patients.
29
Despite these successes, approximately 50% of
patients have intrinsic resistance to tamoxifen
therapy and most of the remainder will acquire
resistance to the drug, leading to metastatic
cancer.
30,31
A further understanding of the
tamoxifen mechanism of action and mechanisms
of intrinsic, or acquired, resistance at the pro-
teome level will aid in the development of modi-
Human Plasma
In an attempt to de
ne the human plasma
proteome, several large projects have been con-
ducted in the last seven years. Typically, the total
number of proteins reported to be identi
ed
directly from serum varies greatly with the
aqueous-phase fractionation methods (e.g.,
SCX) used either individually or in combination
and according to whether antibodies were used
to deplete the most abundant proteins
rst.
Reported numbers range from 252 to approxi-
mately 3,000 proteins depending on statistical
filtering criteria.
23
e
27
When a stringent cutoff
fied disease treatment. As tamoxifen resistance
and agonistic functions have been observed,
we attempted to identify novel proteins upregu-
lated after tamoxifen treatment of MCF-7 breast
cancer cells using the systematic PAcIFIC pro-
teomic screen. Interestingly, the PAcIFIC screen
detected pro-metastatic protein anterior gradient
2 (AGR2) protein as signi
0.95 and no single hits
allowed), the number of identi
filter is applied (p
>
ed proteins is
typically under 200. We note that using the
PAcIFIC method a two- to threefold increase in
proteins identi
cation was observed with less
instrument time and from a sample processed
only through single immuno-depletion step.
12
PAcIFIC also showed some advantage in terms
of dynamic range detected over standard DDA
analysis. When PAcIFIC was compared to the
genome-based DDA GPF method using eight
m/z ranges, PAcIFIC analysis detected proteins
over a dynamic range of eight to mid-nine orders
of magnitude depending on the FDR
cantly upregulated
in tamoxifen-treated cells. However, neither
DDA GPF nor a transcriptomic screen detected
AGR2 protein/transcript at signi
cantly up-
regulated levels. This
finding demonstrates the
importance of increased dynamic range for iden-
tifying unique proteins by PAcIFIC analysis.
Further cell-based experiments using human
cancer cell lines and in vivo xenografts con
filtering
stringency applied. This number represents an
increase of one to three orders of magnitude
(base 10) over genome-based DDA GPF analysis
of the same sample. The most likely explanations
for this increase in dynamic range with PAcIFIC
is that by ignoring detection of the precursor ion,
we do not bias our results toward high abun-
dance signals, which allows PAcIFIC to frag-
ment peptides of very low and no signal/noise.
rmed
the PAcIFIC hypothesis that AGR2 is upregu-
lated in MCF-7 cells post tamoxifen treatment
and that
it
is implicated in drug resistance
mediation.
17
Abdominal Aortic Aneurysm (AAA)
Abdominal aortic aneurysm (AAA) is charac-
terized by increased aortic vessel wall diameter
(
1.5 times normal) and loss of parallelism. This
disease is responsible for 1% to 4% mortality
occurring as a result of acute and unexpected
rupture in males older than 65 years.
32
Due to
>
Breast Cancer
Breast cancer is the most commonly diag-
nosed cancer in women, and it is estimated that
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