Reverse This of course facilitated by proteomic

Reverse phase protein array (RPPA) is a
proteomic assay designed to measure protein expression in a number of samples,
simultaneously. Proteomics is the large scale study of a proteins’ structure,
function and interactions with other proteins. The study of proteins has proven
difficult compared to genomic studies. This because a proteins function,
structure and post translational state fluctuates greatly from cell to cell and
time to time. Quite often post translational modifications (PTMs) are
responsible for determining whether a protein is biologically active or not. Active
proteins, such as phosphorylated kinases, can ignite sequential phosphorylation
of a number of proteins involved in a particular pathway. These signaling pathways
can have a wide range of outcomes and ultimately determine the fate of a cell-
whether it lives or dies. Thus, proteomic studies can be extremely useful in
understanding diseases, their progression and in developing targeted therapies.
Although a wide range of proteomic assays can determine whether a protein is
present or not, RPPA is one of the few that examine the activation state of a
protein. The activation state of a protein can prove vital when studying
multistep diseases such as cancer. Because cancer has both genetic and epigenetic
elements, the functional status of a protein will differ from patient to
patient. Genomic analysis has greatly aided in revealing key oncogenes and
tumor suppressors that have aided in cancer immortality through protein
activation or suppression. However when cancer develops and there are no
genomic susceptibilities, protein deregulation must be explored. For this
reason a lot of cancer research has now shifted focus onto the cancer proteome.
This of course facilitated by proteomic assays, including RPPA (Boja & Rodriguez, 2014).



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RPPA is similar to other immunoassays in
that it uses primary antibodies to probe a sample. The principle is relatively
simple. ‘Reverse phase’ refers to the anylate being immobilized to the solid
surface. A primary antibody (which must undergo a stringent validation process)
is then applied overnight in solution phase. A secondary antibody, conjugated
to an informer (e.g.HRP) is then applies and the signal is detected ‘(Liotta et al., 2003). Typical forward phase assays, such as ELISA, fix the primary
antibody to the solid surface. The sample is then applies inn solution phase
and incubated overnight. This requires a lot of sample which is not viable for
clinical trials whereby the sample can only be obtained via a biopsy. The small
sample required by RPPA is a key advantage over forward phase arrays. The second
primary antibody must also be applied to the sample facilitate secondary
antibody binding. The specificity and cost of two primary antibodies is another
example of how RPPA superseeds forward phase arrays.

RPPA allows for a large multitude of
samples to be tested for specific protein/phosphoprotein under the same experimental
conditions, at the same time. The general procedure can be visualized in figure 1.1. The samples to be analyzed may be cellular lysates (Nishizuka et al., 2003), tissue samples  (Agarwal et al., 2009), or
even bodily fluids such as CSF, serum, urine etc. (Janzi et al., 2009). RPPA can also examine heterogenous cell populations from tissue
samples (Akbani et al., 2014) – Cells grown in culture often have different protein profiles to
cells in a human tissue due to the tissues micro-environment. When developing
drug therapies this needs to be considered. The process of laser capture
microdissection has over-come this problem and can be used in conjunction with
RPPA to obtain a sample that is an accurate representation of cells proteome (Bonner et al., 1997).

Protein extraction is carried out by cell
lysis using denaturing buffers and protease/phosphatase inhibitors. The cell
lysates obtained are generally serially diluted for quantification purposes as
well as quality control. Roughly 1nL of lysate is then printed onto a
nitrocellulose glass slide via an automatic pin based microarrayer. Each slide
is then probed with a primary antibody which can be detected by fluorescent of
chemiluminescent assays. Software then quantifies signal intensities for each




(Spurrier, Ramalingam, & Nishizuka, 2008)



Central to RPPA preparation is antibody
screening. Due to the required sensitivity of antibodies, all must first
undergo a strict validation process. Western blots (WB) are performed using the
same material as used in RPPA. Importantly, antibodies must display a single
band at the appropriate molecular weight for that protein. Antibodies against
phosphorylated proteins must show specificity against stimulated (growth
factors) or inhibited proteins (inhibitors). In many cases antibody
concentration, compared to that used in WB, will be increased in RPPA. Some
antibodies may also need a longer incubation time. The limited number of
sensitive antibodies is RPPAs greatest disadvantage. Guidelines or methods for
antibody validation are not available as of yet however a range of standard
antibodies is available on the MD Anderson cancer webpage.

To examine whether a protein is active or
not, RPPA can be used. The anylate is subjected to two different antibodies,
one directed against the protein (e.g. p70S6K) and one aimed at the
phosphorylated protein (e.g. p70 Ser-394). The difference in levels between
these will allow one to determine the activation state of the protein (Espina, Wulfkuhle, Calvert, Petricoin, & Liotta,