Alkyne-Tag SERS Screening and Identification of Small-Molecule-Binding Sites in Protein
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- Jun Ando
- AMED-CREST, Japan Agency for Medical Research and Development, Saitama 351-0198, Japan
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- Miwako Asanuma
- AMED-CREST, Japan Agency for Medical Research and Development, Saitama 351-0198, Japan
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- Kosuke Dodo
- AMED-CREST, Japan Agency for Medical Research and Development, Saitama 351-0198, Japan
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- Hiroyuki Yamakoshi
- Sodeoka Live Cell Chemistry Project, ERATO, Japan Science and Technology Agency, Saitama 351-0198, Japan
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- Satoshi Kawata
- Department of Applied Physics, Osaka University, Osaka 565-0871, Japan
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- Katsumasa Fujita
- AMED-CREST, Japan Agency for Medical Research and Development, Saitama 351-0198, Japan
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- Mikiko Sodeoka
- AMED-CREST, Japan Agency for Medical Research and Development, Saitama 351-0198, Japan
説明
Identification of small-molecule-binding sites in protein is important for drug discovery and analysis of protein function. Modified amino-acid residue(s) can be identified by proteolytic cleavage followed by liquid chromatography-mass spectrometry (LC-MS), but this is often hindered by the complexity of the peptide mixtures. We have developed alkyne-tag Raman screening (ATRaS) for identifying binding sites. In ATRaS, small molecules are tagged with alkyne and form covalent bond with proteins. After proteolysis and HPLC, fractions containing the labeled peptides with alkyne tags are detected by means of surface-enhanced Raman scattering (SERS) using silver nanoparticles and sent to MS/MS to identify the binding site. The use of SERS realizes high sensitivity (detection limit: ∼100 femtomole) and reproducibility in the peptide screening. By using an automated ATRaS system, we successfully identified the inhibitor-binding site in cysteine protease cathepsin B, a potential drug target and prognostic marker for tumor metastasis. We further showed that the ATRaS system works for complex mixtures of trypsin-digested cell lysate. The ATRaS technology, which provides high molecular selectivity to LC-MS analysis, has potential to contribute in various research fields, such as drug discovery, proteomics, metabolomics and chemical biology.
収録刊行物
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- Journal of the American Chemical Society
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Journal of the American Chemical Society 138 (42), 13901-13910, 2016-10-14
American Chemical Society (ACS)
