US EPA

7420705 

Journal Article 

A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis 

Nury, C; Redeker, V; Dautrey, S; Romieu, A; van Der Rest, G; Renard, PY; Melki, R; Chamot-Rooke, J; , 

2015 

Yes 

Analytical Chemistry
ISSN: 0003-2700
EISSN: 1520-6882 

AMER CHEMICAL SOC 

WASHINGTON 

87 

3 

1853-1860 

English 

25522193 

10.1021/ac503892c 

WOS:000349059000060 

https://pubs.acs.org/doi/10.1021/ac503892c
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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. 

Chromatography; Esterification; Esters; Liquid chromatography; Mass spectrometry; Peptides; Cross-linked proteins; Enzymatic digestions; Interaction analysis; Isotopically labeled; Molecular chaperones; Parkinson's disease; Protein cross-linker; Protein-protein interface; Proteins; alpha synuclein; azide; carbamic acid derivative; cross linking reagent; heat shock cognate protein 70; amino acid sequence; chemical structure; chemistry; human; liquid chromatography; mass spectrometry; metabolism; molecular genetics; Parkinson disease; procedures; protein analysis; protein protein interaction; alpha-Synuclein; Amino Acid Sequence; Azides; Carbamates; Chromatography, Liquid; Cross-Linking Reagents; HSC70 Heat-Shock Proteins; Humans; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Parkinson Disease; Protein Interaction Mapping; Protein Interaction Maps