title: A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis.
abstract: The variety of protein cross-linkers developed in recent years illustrates the current requirement for efficient reagents optimized for mass spectrometry (MS) analysis. To date, the most widely used strategy relies on commercial cross-linkers that bear an isotopically labeled tag and N-hydroxysuccinimid-ester (NHS-ester) moieties. Moreover, an enrichment step using liquid chromatography is usually performed after enzymatic digestion of the cross-linked proteins. Unfortunately, this approach suffers from several limitations. First, it requires large amounts of proteins. Second, NHS-ester cross-linkers are poorly efficient because of their fast hydrolysis in water. Finally, data analysis is complicated because of uneven fragmentation of complex isotopic cross-linked peptide mixtures. We therefore synthesized a new type of trifunctional cross-linker to overrule these limitations. This reagent, named NNP9, comprises a rigid core and bears two activated carbamate moieties and an azido group. NNP9 was used to establish intra- and intermolecular cross-links within creatine kinase, then to map the interaction surfaces between α-Synuclein (α-Syn), the aggregation of which leads to Parkinson's disease, and the molecular chaperone Hsc70. We show that NNP9 cross-linking efficiency is significantly higher than that of NHS-ester commercial cross-linkers. The number of cross-linked peptides identified was increased, and a high quality of MS/MS spectra leading to high sequence coverage was observed. Our data demonstrate the potential of NNP9 for an efficient and straightforward characterization of protein-protein interfaces and illustrate the power of using different cross-linkers to map thoroughly the surface interfaces within protein complexes.
authors.affiliation: Structural Mass Spectrometry and Proteomics Unit, Institut Pasteur, CNRS UMR 3528, Paris, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Nury
authors.forename: Virginie
authors.initials: V
authors.lastname: Redeker
authors.forename: Sébastien
authors.initials: S
authors.lastname: Dautrey
authors.forename: Anthony
authors.initials: A
authors.lastname: Romieu
authors.forename: Guillaume
authors.initials: G
authors.lastname: van der Rest
authors.forename: Pierre-Yves
authors.initials: PY
authors.lastname: Renard
authors.forename: Ronald
authors.initials: R
authors.lastname: Melki
authors.forename: Julia
authors.initials: J
authors.lastname: Chamot-Rooke
mesh_terms: D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry*; D002219:Carbamates / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry*; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010300:Parkinson Disease / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D051844:alpha-Synuclein / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001386:Azides; D002219:Carbamates; D003432:Cross-Linking Reagents; D050883:HSC70 Heat-Shock Proteins; D051844:alpha-Synuclein