title: A highly efficient multifunctional tandem affinity purification approach applicable to diverse organisms.
abstract: 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.
authors.affiliation: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
authors.forename: Hanhui
authors.initials: H
authors.lastname: Ma
authors.forename: Janel R
authors.initials: JR
authors.lastname: McLean
authors.forename: Lucy Fang-I
authors.initials: LF
authors.lastname: Chao
authors.forename: Sebastian
authors.initials: S
authors.lastname: Mana-Capelli
authors.forename: Murugan
authors.initials: M
authors.lastname: Paramasivam
authors.forename: Kirsten A
authors.initials: KA
authors.lastname: Hagstrom
authors.forename: Kathleen L
authors.initials: KL
authors.lastname: Gould
authors.forename: Dannel
authors.initials: D
authors.lastname: McCollum
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D029742:Caenorhabditis elegans Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins