A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis.
Anal Chem
; 87(3): 1853-60, 2015 Feb 03.
Article
em En
| MEDLINE
| ID: mdl-25522193
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.
Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Azidas
/
Carbamatos
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Reagentes de Ligações Cruzadas
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Mapeamento de Interação de Proteínas
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Proteínas de Choque Térmico HSC70
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Alfa-Sinucleína
Limite:
Humans
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article