Discovery of Cell Compartment Specific Protein–Protein Interactions using Affinity Purification Combined with Tandem Mass Spectrometry

Affinity purification combined with tandem mass spectrometry
(AP-MS/MS)
is a well-established method used to discover interaction partners
for a given protein of interest. Because most AP-MS/MS approaches
are performed using the soluble fraction of whole cell extracts (WCE),
information about the cellular compartments where the interactions
occur is lost. More importantly, classical AP-MS/MS often fails to
identify interactions that take place in the nonsoluble fraction of
the cell, for example, on the chromatin or membranes; consequently,
protein complexes that are less soluble are underrepresented. In this
paper, we introduce a method called multiple cell compartment AP-MS/MS
(MCC-AP-MS/MS), which identifies the interactions of a protein independently
in three fractions of the cell: the cytoplasm, the nucleoplasm, and
the chromatin. We show that this fractionation improves the sensitivity
of the method when compared to the classical affinity purification
procedure using soluble WCE while keeping a very high specificity.
Using three proteins known to localize in various cell compartments
as baits, the CDK9 subunit of transcription elongation factor P-TEFb,
the RNA polymerase II (RNAP II)-associated protein 4 (RPAP4), and
the largest subunit of RNAP II, POLR2A, we show that MCC-AP-MS/MS
reproducibly yields fraction-specific interactions. Finally, we demonstrate
that this improvement in sensitivity leads to the discovery of novel
interactions of RNAP II carboxyl-terminal domain (CTD) interacting
domain (CID) proteins with POLR2A.