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Molecular Epitope Determination of Aptamer Complexes of the Multidomain Protein C‐Met by Proteolytic Affinity‐Mass Spectrometry
ChemMedChem ( IF 3.016 ) Pub Date : 2020-01-16 , DOI: 10.1002/cmdc.201900489
Loredana Lupu; Pascal Wiegand; Nico Hüttmann; Stephan Rawer; Wolfgang Kleinekofort; Irina Shugureva; Anna S. Kichkailo; Felix N. Tomilin; Alexander Lazarev; Maxim V. Berezovski; Michael Przybylski

C‐Met protein is a glycosylated receptor tyrosine kinase of the hepatocyte growth factor (HGF), composed of an α and a β chain. Upon ligand binding, C‐Met transmits intracellular signals by a unique multi‐substrate docking site. C‐Met can be aberrantly activated leading to tumorigenesis and other diseases, and has been recognized as a biomarker in cancer diagnosis. C‐Met aptamers have been recently considered a useful tool for detection of cancer biomarkers. Herein we report a molecular interaction study of human C‐Met expressed in kidney cells with two DNA aptamers of 60 and 64 bases (CLN0003 and CLN0004), obtained using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure. Epitope peptides of aptamer‐C‐Met complexes were identified by proteolytic affinity‐mass spectrometry in combination with SPR biosensor analysis (PROTEX‐SPR‐MS), using high‐pressure proteolysis for efficient digestion. High affinities (KD, 80–510 nM) were determined for aptamer‐C‐Met complexes, with two‐step binding suggested by kinetic analysis. A linear epitope, C‐Met (381–393) was identified for CLN0004, while the CLN0003 aptamer revealed an assembled epitope comprised of two peptide sequences, C‐Met (524–543) and C‐Met (557–568). Structure modeling of C‐Met‐aptamers were consistent with the identified epitopes. Specificities and affinities were ascertained by SPR analysis of the synthetic epitope peptides. The high affinities of aptamers to C‐Met, and the specific epitopes revealed render them of high interest for cellular diagnostic studies.
更新日期:2020-01-17

 

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