title: Sortase A mediated site-specific immobilization for identification of protein interactions in affinity purification-mass spectrometry experiments.
abstract: Proteomics approaches using MS in combination with affinity purification have emerged as powerful tools to study protein-protein interactions. Here we make use of the specificity of sortase A transpeptidation reaction to prepare affinity matrices in which a protein bait is covalently linked to the matrix via a short C-terminal linker region. As a result of this site-directed immobilization, the bait remains functionally accessible to protein interactions. To apply this approach, we performed SILAC-based pull-down experiments and demonstrate the suitability of the approach.
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany; Institut für Chemie und Biochemie, Freie Universität Berlin, Berlin, Germany.
authors.forename: Benno
authors.initials: B
authors.lastname: Kuropka
authors.forename: Nadine
authors.initials: N
authors.lastname: Royla
authors.forename: Christian
authors.initials: C
authors.lastname: Freund
authors.forename: Eberhard
authors.initials: E
authors.lastname: Krause
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006801:Humans; D019169:Jurkat Cells; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
keywords: Mass spectrometry; Protein interactions; SILAC; Sortase A; Technology