Biology Reference
In-Depth Information
a suitable antibody against the targeted endog-
enous protein that can be used for the pulldown
is a common problem that often prevents the
complex isolation from being carried out under
normal physiological conditions.
Prior to LC-MS
2
analysis, fractionation of the
cant effort is now under way using
different approaches
A signi
to minimize the false
discovery rate in af
cation experi-
ments.
67
There are many factors that affect the
quality of these experiments, such as the af
nity puri
nity
resin (beads), protein concentration during the
immunoprecipitation, cell type, lysis buffer,
incubation times, wash conditions, fractionation
techniques, and the MS
2
capabilities. It is there-
fore imperative to carefully plan these experi-
ments to minimize the false discovery.
A highly explored technique to address back-
ground proteins in af
af
ed intact proteins using SDS-
PAGE or peptides produced by tryptically
digested the complex using methods such as
strong cationic exchange (SCX) has been demon-
strated to increase the number of
nity puri
ed
proteins.
63
A side-by-side comparison of these
two methods has demonstrated that fraction-
ation by SCX signi
identi
cation experi-
ments is the construction of a bead proteome
database, which in theory should include all
proteins capable of binding the matrix material
and identi
nity puri
cantly increases the number
of
ed proteins and individual protein
coverage following af
identi
cation, particu-
larly for target proteins expressed at low levels.
64
However, it should be noted that even though
these methods increase the number of proteins
identi
nity puri
ed by the mass spectrometer.
68
Once these proteins have been identi
ed, they
can be used to
filter the actual experimental
data for background proteins. The alternative is
to construct a database of proteins from all avail-
able af
ed and therefore the likelihood of identi-
fying a potential interacting proteins, they will
also increase the number of nonspeci
c back-
ground proteins detected. Another source of
false negative discovery is the data-dependent
acquisition method commonly used during
MS
2
identi
cations and use it to identify
proteins that are pulled down with multiple
different baits.
69
Any protein that is frequently
identi
nity puri
c
background protein. The drawback to this
method is that frequently an af
ed has the potential to be a nonspeci
cation.
65
Therefore, although af
nity
puri
cantly reduces the complexity
of the sample, a mass spectrometer with fast
scanning time is highly preferable when search-
ing for novel protein interactions.
cation signi
cation
study is carried out with several proteins associ-
ated with the same biological process. As it
would be expected that some of the interacting
protein(s) will associate with several of the bait
proteins,
nity puri
filtering method could lead to
a high false negative discovery rate.
Another approach used to identify interacting
proteins is stable isotope labeling by amino acids
in cell culture (SILAC).
70
In the simplest varia-
tion of this method, cells expressing the protein
of interest are grown in culture media that uses
a
this
ADDRESSING THE BACKGROUND
PROBLEM
cant effort has been exerted to address
the limitations of the two dominant high-
throughput protein interaction discovery
methods. As mentioned previously, several
novel techniques that utilize the same principle
as the classical yeast 2-hybrid method have
been developed and the construction of nonspe-
ci
Signi
isotope of an amino acid(s) as the
control cell lines use the
“
heavy
”
“
light
”
isotope. After
performing two separate af
cation
experiments, the samples are mixed at a 1:1 ratio
prior to analysis. The mass difference resulting
from the use of these different heavy and light
nity puri
c protein interaction databases can aid in the
minimization of false positives discovery .
66
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