A Highly Efficient Multifunctional Tandem Affinity Purification Approach Applicable to Diverse Organisms

被引:30
作者
Ma, Hanhui [1 ,2 ]
McLean, Janel R. [3 ,4 ]
Chao, Lucy Fang-I [2 ,5 ]
Mana-Capelli, Sebastian [1 ,2 ]
Paramasivam, Murugan [1 ,2 ]
Hagstrom, Kirsten A. [2 ,5 ]
Gould, Kathleen L. [3 ,4 ]
McCollum, Dannel [1 ,2 ]
机构
[1] Univ Massachusetts, Sch Med, Dept Microbiol & Physiol Syst, Worcester, MA 01605 USA
[2] Univ Massachusetts, Sch Med, Program Cell Dynam, Worcester, MA 01605 USA
[3] Vanderbilt Univ, Howard Hughes Med Inst, Sch Med, Nashville, TN 37232 USA
[4] Vanderbilt Univ, Dept Cell & Dev Biol, Sch Med, Nashville, TN 37232 USA
[5] Univ Massachusetts, Sch Med, Dept Mol Med, Worcester, MA 01605 USA
基金
美国国家卫生研究院;
关键词
GREEN FLUORESCENT PROTEIN; QUANTITATIVE PHOSPHOPROTEOMICS; PEPTIDE IDENTIFICATION; PLK1; GENE; PHOSPHORYLATION; AURORA; ACTIVATION; EXPRESSION; PROTEOMICS;
D O I
10.1074/mcp.O111.016246
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Determining the localization, binding partners, and secondary modifications of individual proteins is crucial for understanding protein function. Several tags have been constructed for protein localization or purification under either native or denaturing conditions, but few tags permit all three simultaneously. Here, we describe a multifunctional tandem affinity purification (MAP) method that is both highly efficient and enables protein visualization. The MAP tag utilizes affinity tags inserted into an exposed surface loop of mVenus offering two advantages: (1) mVenus fluorescence can be used for protein localization or FACS-based selection of cell lines; and (2) spatial separation of the affinity tags from the protein results in high recovery and reduced variability between proteins. MAP purification was highly efficient in multiple organisms for all proteins tested. As a test case, MAP combined with liquid chromatography-tandem MS identified known and new candidate binding partners and modifications of the kinase Plk1. Thus the MAP tag is a new powerful tool for determining protein modification, localization, and interactions. Molecular & Cellular Proteomics 11: 10.1074/mcp.O111.016246, 501-511, 2012.
引用
收藏
页码:501 / 511
页数:11
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