title: PUB-MS: a mass spectrometry-based method to monitor protein-protein proximity in vivo.
abstract: The common techniques to study protein-protein proximity in vivo are not well adapted to the capabilities and the expertise of a standard proteomics laboratory, typically based on the use of mass spectrometry. With the aim of closing this gap, we have developed PUB-MS (for proximity utilizing biotinylation and mass spectrometry), an approach to monitor protein-protein proximity, based on biotinylation of a protein fused to a biotin-acceptor peptide (BAP) by a biotin-ligase, BirA, fused to its interaction partner. The biotinylation status of the BAP can be further detected by either Western analysis or mass spectrometry. The BAP sequence was redesigned for easy monitoring of the biotinylation status by LC-MS/MS. In several experimental models, we demonstrate that the biotinylation in vivo is specifically enhanced when the BAP- and BirA-fused proteins are in proximity to each other. The advantage of mass spectrometry is demonstrated by using BAPs with different sequences in a single experiment (allowing multiplex analysis) and by the use of stable isotopes. Finally, we show that our methodology can be also used to study a specific subfraction of a protein of interest that was in proximity with another protein at a predefined time before the analysis.
authors.affiliation: Institut Gustave Roussy , 39 Rue Camilles Desmoulin, 94805, Villejuif, France.
authors.forename: Arman
authors.initials: A
authors.lastname: Kulyyassov
authors.forename: Muhammad
authors.initials: M
authors.lastname: Shoaib
authors.forename: Andrei
authors.initials: A
authors.lastname: Pichugin
authors.forename: Patricia
authors.initials: P
authors.lastname: Kannouche
authors.forename: Erlan
authors.initials: E
authors.lastname: Ramanculov
authors.forename: Marc
authors.initials: M
authors.lastname: Lipinski
authors.forename: Vasily
authors.initials: V
authors.lastname: Ogryzko
mesh_terms: D000595:Amino Acid Sequence; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D005347:Fibroblasts / Q000166:cytology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D007554:Isotopes / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D007554:Isotopes; D011994:Recombinant Proteins; D001710:Biotin