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Molecular Epitope Determination of Aptamer Complexes of the Multidomain Protein C-Met by Proteolytic Affinity-Mass Spectrometry.
ChemMedChem ( IF 3.6 ) Pub Date : 2019-12-11 , DOI: 10.1002/cmdc.201900489 Loredana Lupu 1 , Pascal Wiegand 1 , Nico Hüttmann 1, 2 , Stephan Rawer 1 , Wolfgang Kleinekofort 1, 3 , Irina Shugureva 4, 5 , Anna S Kichkailo 5 , Felix N Tomilin 4, 6 , Alexander Lazarev 7 , Maxim V Berezovski 2 , Michael Przybylski 1
ChemMedChem ( IF 3.6 ) Pub Date : 2019-12-11 , DOI: 10.1002/cmdc.201900489 Loredana Lupu 1 , Pascal Wiegand 1 , Nico Hüttmann 1, 2 , Stephan Rawer 1 , Wolfgang Kleinekofort 1, 3 , Irina Shugureva 4, 5 , Anna S Kichkailo 5 , Felix N Tomilin 4, 6 , Alexander Lazarev 7 , Maxim V Berezovski 2 , Michael Przybylski 1
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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.
中文翻译：
通过蛋白水解亲和质谱法测定多域蛋白C-Met的适体复合物的分子表位。
C-Met蛋白是肝细胞生长因子（HGF）的糖基化受体酪氨酸激酶，由α和β链组成。配体结合后，C-Met通过独特的多底物对接位点传输细胞内信号。C-Met可能被异常激活，从而导致肿瘤发生和其他疾病，并且已被公认为癌症诊断中的生物标记。最近，C-Met适体被认为是检测癌症生物标志物的有用工具。本文中，我们报道了使用SELEX（通过指数富集进行配体的系统进化）方法获得的具有60和64个碱基的两个DNA适体（CLN0003和CLN0004）在肾细胞中表达的人C-Met的分子相互作用的研究。通过高压蛋白水解有效消化，通过蛋白水解亲和质谱结合SPR生物传感器分析（PROTEX-SPR-MS）鉴定了适体-C-Met复合物的表位肽。确定了适体-C-Met复合物的高亲和力（KD，80-510 nM），通过动力学分析建议了两步结合。对于CLN0004，鉴定了线性表位C-Met（381-393），而CLN0003适体显示了由两个肽序列C-Met（524-543）和C-Met（557-568）组成的组装表位。C-Met-适体的结构模型与所鉴定的表位一致。通过合成表位肽的SPR分析确定特异性和亲和力。适体对C-Met的高度亲和力以及所揭示的特定表位使其成为细胞诊断研究的高度关注。
更新日期：2020-01-17
中文翻译：
通过蛋白水解亲和质谱法测定多域蛋白C-Met的适体复合物的分子表位。
C-Met蛋白是肝细胞生长因子（HGF）的糖基化受体酪氨酸激酶，由α和β链组成。配体结合后，C-Met通过独特的多底物对接位点传输细胞内信号。C-Met可能被异常激活，从而导致肿瘤发生和其他疾病，并且已被公认为癌症诊断中的生物标记。最近，C-Met适体被认为是检测癌症生物标志物的有用工具。本文中，我们报道了使用SELEX（通过指数富集进行配体的系统进化）方法获得的具有60和64个碱基的两个DNA适体（CLN0003和CLN0004）在肾细胞中表达的人C-Met的分子相互作用的研究。通过高压蛋白水解有效消化，通过蛋白水解亲和质谱结合SPR生物传感器分析（PROTEX-SPR-MS）鉴定了适体-C-Met复合物的表位肽。确定了适体-C-Met复合物的高亲和力（KD，80-510 nM），通过动力学分析建议了两步结合。对于CLN0004，鉴定了线性表位C-Met（381-393），而CLN0003适体显示了由两个肽序列C-Met（524-543）和C-Met（557-568）组成的组装表位。C-Met-适体的结构模型与所鉴定的表位一致。通过合成表位肽的SPR分析确定特异性和亲和力。适体对C-Met的高度亲和力以及所揭示的特定表位使其成为细胞诊断研究的高度关注。
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