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Interactomics - Coggle Diagram
Interactomics
Techniques to study protein-protein interactions
Tandem Affinity Purification Tagging
(
TAP
)
System which allows to identify and isolate proteins interacting with a target (
bait
)
Tag structure
Bait
Calmodulin binding peptide
TEV protease cleavage site
Protein A
Affinity chromatography with IgG beads
Cleavage by TEV
Binding to calmodulin beads
Interacts with proteins to be identified
Workflow:
Incubation of the tag with proteins. Some will interact and bind to the bait
The complex interactors-bait-tag will bind to
IgG beads
(1st affinity column). Some contaminants may also bind
Cleavage with
TEV protease
to release the interactors-bait-calmodulin-binding peptide complex
The complex binds to
calmodulin beads
(2nd affinity column)
Elution of bait and interactors
The isolation of interactors can be followed by
LC/MS
to identify them
Can be used during
SILAC
experiments
Contrary to normal SILAC experiments, samples are
kept separate during the TAP purification process
and
only mixed after
. This is done to
avoid contamination
. Otherwise, we'd lose the ability to distinguish which interactors came from which condition because both baits would capture proteins from the mixed lysate.
Used for the identification of
yeast protein interactome
, revealing a complex network of
highly interconnected proteins
Proximity-Dependent Biotinylation
Tag structure:
Bait
BirA enzyme
Catalyzes the reaction of
in situ
biotinylation
of proteins which are in close proximity of the bait (within
10 nm
) and supposedely interact with it
Interactors can be purified using
streptavidin beads
Crosslinking-based interactomics
Provides information on the
structure
of the target protein and its interactors
Makes use of a crosslinking agent which creases cross-links within the target protein (
intera-protein cross-links
) and the complex with the interactors (
inter-protein cross links
)
Workflow:
Cross-linking
Digestion of the protein complexes with proteases to obtain peptides
Enrichment of cross-linked peptides with cation exchange chromatography
MS
STRING
Database for protein-protein interactions networks
data comes from experimental studies, data mining, evolutionary studies
