Identifying Novel Protein Complexes in Cancer Cells Using Epitope-Tagging of Endogenous Human Genes and Affinity-Purification Mass Spectrometry

Affinity-purification mass spectrometry (AP-MS) is the
preeminent
technique for identification of eukaryotic protein complexes in vivo. AP-MS workflows typically express epitope-tagged
bait proteins, immunopurify, and then identify associated protein
complexes using mass spectrometry. However, challenges of existing
strategies include the construction of expression vectors for large
open reading frames and the possibility that overexpression of bait
proteins may result in expression of nonphysiological levels of the
bait protein with concomitant perturbation of endogenous protein complexes.
To address these issues, we use human cell lines with epitope-tagged
endogenous genes as AP-MS substrates to develop a platform that we
call "knock-in AP-MS", thereby avoiding the challenges of expression
vector construction and ensuring that expression of tagged proteins
is driven by endogenous regulatory mechanisms. Using three different
bait genes (MRE11A, DNMT1 and APC), we show that cell lines expressing
epitope-tagged endogenous genes make good substrates for sensitive
and reproducible identification of protein interactions using AP-MS.
In particular, we identify novel interactors of the important oncoprotein
Adenomatous Polyposis Coli (APC), including an interaction with Flightless-1
homologue (FLII) that is enriched in nuclear fractions.