title: Mapping a noncovalent protein-peptide interface by top-down FTICR mass spectrometry using electron capture dissociation.
abstract: Noncovalent protein-ligand and protein-protein complexes are readily detected using electrospray ionization mass spectrometry (ESI MS). Furthermore, recent reports have demonstrated that careful use of electron capture dissociation (ECD) fragmentation allows covalent backbone bonds of protein complexes to be dissociated without disruption of noncovalent protein-ligand interactions. In this way the site of protein-ligand interfaces can be identified. To date, protein-ligand complexes, which have proven tractable to this technique, have been mediated by ionic electrostatic interactions, i.e., ion pair interactions or salt bridging. Here we extend this methodology by applying ECD to study a protein-peptide complex that contains no electrostatics interactions. We analyzed the complex between the 21 kDa p53-inhibitor protein anterior gradient-2 and its hexapeptide binding ligand (PTTIYY). ECD fragmentation of the 1:1 complex occurs with retention of protein-peptide binding and analysis of the resulting fragments allows the binding interface to be localized to a C-terminal region between residues 109 and 175. These finding are supported by a solution-phase competition assay, which implicates the region between residues 108 and 122 within AGR2 as the PTTIYY binding interface. Our study expands previous findings by demonstrating that top-down ECD mass spectrometry can be used to determine directly the sites of peptide-protein interfaces. This highlights the growing potential of using ECD and related top-down fragmentation techniques for interrogation of protein-protein interfaces.
authors.affiliation: SIRCAMS, EastChem School of Chemistry, University of Edinburgh, Edinburgh, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Clarke
authors.forename: Euan
authors.initials: E
authors.lastname: Murray
authors.forename: Ted
authors.initials: T
authors.lastname: Hupp
authors.forename: C Logan
authors.initials: CL
authors.lastname: Mackay
authors.forename: Pat R R
authors.initials: PR
authors.lastname: Langridge-Smith
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004583:Electrons; D005583:Fourier Analysis; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009088:Mucoproteins; D015513:Oncogene Proteins; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C502296:AGR2 protein, human; D009088:Mucoproteins; D015513:Oncogene Proteins; D010446:Peptide Fragments; D011506:Proteins