title: Identifying novel protein complexes in cancer cells using epitope-tagging of endogenous human genes and affinity-purification mass spectrometry.
abstract: 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.
authors.affiliation: Center for Proteomics and Bioinformatics, Department of Genetics and Genome Science, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, United States.
authors.forename: Jing
authors.initials: J
authors.lastname: Song
authors.forename: Yujun
authors.initials: Y
authors.lastname: Hao
authors.forename: Zhanwen
authors.initials: Z
authors.lastname: Du
authors.forename: Zhenghe
authors.initials: Z
authors.lastname: Wang
authors.forename: Rob M
authors.initials: RM
authors.lastname: Ewing
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000302:isolation & purification / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism / Q000473:pathology; D002846:Chromatography, Affinity / Q000379:methods*; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology*; D003593:Cytoplasm / Q000378:metabolism / Q000473:pathology; D000229:Dependovirus; D018604:Epitope Mapping / Q000379:methods*; D005822:Genetic Vectors; D015894:Genome, Human; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008840:Microfilament Proteins / Q000302:isolation & purification / Q000378:metabolism; D046912:Multiprotein Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods; D040901:Proteomics / Q000379:methods; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015534:Trans-Activators; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C413364:ERBIN protein, human; C098179:FLII protein, human; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D009363:Neoplasm Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D015534:Trans-Activators