title: A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis.
abstract: Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University.
authors.forename: Megumi
authors.initials: M
authors.lastname: Tsuchiya
authors.affiliation: Department of Biotechnology and Genetic Engineering, Jahangirnagar University.
authors.forename: M Rezaul
authors.initials: MR
authors.lastname: Karim
authors.affiliation: Division of Cell Regeneration and Transplantation, School of Medicine, Nihon University.
authors.forename: Taro
authors.initials: T
authors.lastname: Matsumoto
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University; hidesato@fbs.osaka-u.ac.jp.
authors.forename: Hidesato
authors.initials: H
authors.lastname: Ogawa
authors.affiliation: Division of Cell Regeneration and Transplantation, School of Medicine, Nihon University; Institute of Genetics and Animal Breeding of the Polish Academy of Sciences; Graduate School of Life and Medical Sciences, Doshisha University; hitanigu@mail.doshisha.ac.jp.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Taniguchi
mesh_terms: D002843:Chromatin / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D004265:DNA Helicases / Q000378:metabolism*; D057809:HEK293 Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D060066:Protein Interaction Maps; D011506:Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D002843:Chromatin; D006657:Histones; D011506:Proteins; D014157:Transcription Factors; D004265:DNA Helicases; C546908:RAD54L2 protein, human