Center for Proteomics and Bioinformatics, Department of Genetics and Genome Science, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, United States.
J Proteome Res. 2012 Dec 7;11(12):5630-41. doi: 10.1021/pr300598t. Epub 2012 Nov 7.
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.
亲和纯化质谱法 (AP-MS) 是鉴定真核生物体内蛋白质复合物的卓越技术。AP-MS 工作流程通常表达表位标记的诱饵蛋白，免疫纯化，然后使用质谱鉴定相关的蛋白质复合物。然而，现有策略的挑战包括构建大片段开放阅读框的表达载体，以及诱饵蛋白过表达可能导致非生理水平的诱饵蛋白表达，从而伴随内源性蛋白质复合物的扰动。为了解决这些问题，我们使用带有表位标记的内源性基因的人细胞系作为 AP-MS 底物，开发了一种我们称之为“敲入 AP-MS”的平台，从而避免了表达载体构建的挑战，并确保标记蛋白的表达受内源性调节机制的驱动。使用三个不同的诱饵基因 (MRE11A、DNMT1 和 APC)，我们表明表达表位标记内源性基因的细胞系是使用 AP-MS 进行敏感和可重复的蛋白质相互作用鉴定的良好底物。特别是，我们鉴定了重要致癌蛋白腺瘤性结肠息肉病 (APC) 的新相互作用因子，包括与核分数中富集的飞行无翅同源物 (FLII) 的相互作用。
