Biology Reference
In-Depth Information
A: Affinity purification - mass spectrometry workflow
Inter-
actor
specific interactors
IP
bait
tag
AB
back-
ground
quantitative
mass spectormetry
control IP
ratio
0
AB
back-
ground
background binders
B: Interaction topology
C: Interaction networks
baits
A
BCDE
A
chemical
crosslinker
digestion
B
C
D
E
extract interactors
from large-scale
AP-MS dataset
identify
cross-linked
peptides
build
topological
model
A
C
D
construct
interaction
network
B
E
FIGURE 1.4
Interaction proteomics. A: Generic scheme of the affinity purification
mass spectrometry workflow. Quantitative comparison of the
amounts of proteins in affinity purifications vs. control purifications distinguishes specific interactors from background binders. B: Protein complexes
treated with chemical cross-linkers. The identification of cross-linked peptides yields spatial restraints that can be used to infer the topology of interactions
and to map binding sites. C: Construction of interaction networks from large-scale AP-MS datasets.
e
'Chemical proteomics' approaches make use of
immobilized small molecule inhibitors to capture and
identify their cellular binding proteins
[119]
. Although this
constitutes a powerful and generic approach, synthesizing
a suitable, immobilizable derivative of an individual small
molecule of interest can be challenging and in some cases
impossible. Alternatively, broadly selective inhibitors can
be used for affinity-capture of a target protein class. This
has been successfully applied for profiling inhibitors tar-
geting kinases
[120,121]
and more recently histone
deacetylases
[122]
. Inhibitor affinity towards its binding
partners can be measured by quantitative, dose-dependent
assays by monitoring the binding response to different
concentrations of the free molecule. Quantitative drug
affinity purification experiments thereby provide a concep-
tual framework for identifying the protein targets that
mediate drug responsiveness and those that potentially
cause side effects. Proteome-wide determination of drug
targets may also reveal alternate therapeutic uses.
Quantitative MS-based approaches furthermore enable
researchers to determine the proteomes of subcellular
structures or organelles, which can only be enriched from
a whole-cell preparation, but not be purified biochemically
[123,124]
. One principle is to profile proteins along
gradients or across different enrichment steps and to clas-
sify them by correlation to known marker proteins. This
approach
was used
to assign proteins to their respective compartments
[125]
,
e
termed protein correlation profiling
e
