A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis.
Identifieur interne : 000724 ( Main/Exploration ); précédent : 000723; suivant : 000725A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis.
Auteurs : Catherine Nury [France] ; Virginie Redeker ; Sébastien Dautrey ; Anthony Romieu ; Guillaume Van Der Rest ; Pierre-Yves Renard ; Ronald Melki ; Julia Chamot-RookeSource :
- Analytical chemistry [ 1520-6882 ] ; 2015.
English descriptors
- KwdEn :
- Amino Acid Sequence, Azides (chemistry), Carbamates (chemistry), Chromatography, Liquid (methods), Cross-Linking Reagents (chemistry), HSC70 Heat-Shock Proteins (chemistry), HSC70 Heat-Shock Proteins (metabolism), Humans, Mass Spectrometry (methods), Models, Molecular, Molecular Sequence Data, Parkinson Disease (metabolism), Protein Interaction Mapping (methods), Protein Interaction Maps, alpha-Synuclein (chemistry), alpha-Synuclein (metabolism).
- MESH :
- chemical , chemistry : Azides, Carbamates, Cross-Linking Reagents, HSC70 Heat-Shock Proteins, alpha-Synuclein.
- chemical , metabolism : HSC70 Heat-Shock Proteins, alpha-Synuclein.
- metabolism : Parkinson Disease.
- methods : Chromatography, Liquid, Mass Spectrometry, Protein Interaction Mapping.
- Amino Acid Sequence, Humans, Models, Molecular, Molecular Sequence Data, Protein Interaction Maps.
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.
DOI: 10.1021/ac503892c
PubMed: 25522193
Affiliations:
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Le document en format XML
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<term>Cross-Linking Reagents (chemistry)</term>
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<term>Models, Molecular</term>
<term>Molecular Sequence Data</term>
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<term>Protein Interaction Maps</term>
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<term>alpha-Synuclein</term>
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<term>Protein Interaction Mapping</term>
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<term>Molecular Sequence Data</term>
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<front><div type="abstract" xml:lang="en">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.</div>
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