Identifying Novel Protein Complexes in Cancer Cells Using Epitope-Tagging of Endogenous Human Genes and Affinity-Purification Mass Spectrometry
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- 作者：Jing Song ; Yujun Hao ; Zhanwen Du ; Zhenghe Wang ; Rob M. Ewing
- 刊名：Journal of Proteome Research
- 出版年：2012
- 出版时间：December 7, 2012
- 年：2012
- 卷：11
- 期：12
- 页码：5630-5641
- 全文大小：474K
- 年卷期：v.11,no.12(December 7, 2012)
- ISSN：1535-3907
文摘
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.