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Identifying novel protein complexes in cancer cells using epitope-tagging of endogenous human genes and affinity-purification mass spectrometry

Identifying novel protein complexes in cancer cells using epitope-tagging of endogenous human genes and affinity-purification mass spectrometry
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.
1535-3893
5630-5641
Song, Jing
c3f6ccf2-4c63-487c-9c39-e25382b9ee91
Hao, Yujun
d77fcc30-ae22-4cd2-8b43-a5ecd089b751
Du, Zhanwen
78a06095-e02b-4461-aca6-562ddd99bf84
Wang, Zhenghe
9142416c-1981-4fa6-9fd7-e0a61a81fc33
Ewing, Rob M.
022c5b04-da20-4e55-8088-44d0dc9935ae
Song, Jing
c3f6ccf2-4c63-487c-9c39-e25382b9ee91
Hao, Yujun
d77fcc30-ae22-4cd2-8b43-a5ecd089b751
Du, Zhanwen
78a06095-e02b-4461-aca6-562ddd99bf84
Wang, Zhenghe
9142416c-1981-4fa6-9fd7-e0a61a81fc33
Ewing, Rob M.
022c5b04-da20-4e55-8088-44d0dc9935ae

Song, Jing, Hao, Yujun, Du, Zhanwen, Wang, Zhenghe and Ewing, Rob M. (2012) Identifying novel protein complexes in cancer cells using epitope-tagging of endogenous human genes and affinity-purification mass spectrometry. Journal of Proteome Research, 11 (12), 5630-5641. (doi:10.1021/pr300598t). (PMID:23106643)

Record type: Article

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.

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Published date: 7 December 2012
Organisations: Molecular and Cellular, Biomedicine, Centre for Biological Sciences

Identifiers

Local EPrints ID: 348398
URI: http://eprints.soton.ac.uk/id/eprint/348398
ISSN: 1535-3893
PURE UUID: 92a35062-9651-4687-9ff1-e965a1899f83
ORCID for Rob M. Ewing: ORCID iD orcid.org/0000-0001-6510-4001

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Date deposited: 13 Feb 2013 11:33
Last modified: 10 Dec 2019 01:36

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Contributors

Author: Jing Song
Author: Yujun Hao
Author: Zhanwen Du
Author: Zhenghe Wang
Author: Rob M. Ewing ORCID iD

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