A Role for ProteineProtein Interaction Networks in the Identification and Characterization of Potential Biomarkers - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 333

the proteins to be probed or between the fused
subunits and their functional partners. Also,
many proteins require post-translational modifi-

membrane protein interactions due to hydro-
phobic residues being exposed when they would
normally be buried in a membrane.
It has been estimated that the accuracy of
these high-throughput yeast 2-hybrid methods
may be less than 10%. 60 Another problem is the
lack of reproducibility between independent
yeast two-hybrid studies of the same bait
protein. A study performed by Ito et al. 61
showed that only 30% of the interactions were
observed between two studies utilizing the
same yeast 2-hybrid method.

-
cations prior to proper function, which would
presumably be different or completely lacking
in a yeast system as compared to higher eukary-
otes. Furthermore, transient interactions, such as
proteins with enzymatic activity, are especially
dif cult to detect due to the short time frame in

which they occur. These factors all contribute
to the false negative rate observed in the yeast
2-hybrid system.
The occurrence of false positives can be even
more problematic than the false negatives
because these results either lead to fruitless vali-
dation efforts or get reported in bulk form and
are propagated throughout the scienti

Af nity Puri cation

cation studies, cell lysate con-
taining the protein of interest is incubated in the
presence of an af

In af nity puri

c knowl-
edge base. There are several causes for a yeast
2-hybrid method to indicate that two proteins
associate when this interaction does not occur
naturally. For example the proteins are overex-
pressed and forced to locate to the nucleus,
which may cause two proteins to interact that
would never encounter each other in their native
locations within the cell. As mentioned previ-
ously, some proteins (such as transcription
factors) may act as self-activators of transcription
or a protein can interact with one of the fusion
domains on the complementary construct in
any of the other permutations of yeast 2-hybrid
method. However, the cause that is probably
the most prevalent is nonspeci

nity reagent, typically an anti-
body and a resin that is capable of precipitating
the protein of interest along with any interacting
proteins. The primary advantage of this method
is that the protein of interest is af

ed
from a cell lysate that contains potentially all of
the interacting partners leading to isolation of
the entire protein complex in a single experi-
ment. However, as for the yeast 2-hybrid system,
the primary challenge during af

nity puri

ca-
tion is to minimize both the false positive and
false negative discovery rate.
The format

nity puri

that is most frequently used

for high-throughput af cation, and

therefore accounts for the majority of the
protein e protein interactions reported in the data-
bases, is to express a tagged version of the protein
of interest in heterologous cells such as HEK293T
cells. There are several inherent factors with this
experimental setup that are thought to account
for much of the false positive and negative
discovery rates. For example, a signi

nity puri

c interactions.
The creation of a list of these nonspeci c interac-

tions can be helpful when selecting candidates
for validation of potential interacting proteins.
Other factors that contribute to false discovery
in yeast 2-hybrid experiments include the utiliza-
tion of protein fragments instead of intact
proteins, the binary nature of the interactions,
the limitations in library construction, screening
that precludes the possibility of screening every
potential binary interaction in a high-throughput
study, the

cant overex-
pression of the protein of interest, compared to the
endogenous level, can lead to forced association
between proteins that do not interact under phys-
iological concentrations. This situation might be
exacerbatedwhen the whole cell is lysed in prepa-
ration for the af

fixed orientation of the protein frag-
ments relative to each other, and the translational
machinery they are fused to, and the bias toward nity puri cation, leading to