Electrochemically-Enhanced Plasmonic Imaging for Quantitative Proteomics

描述(由申请人提供)：我们正在提出一项技术，以帮助蛋白质组学的三个关键领域，包括(A)识别蛋白质相互作用，(B)POST的特征 (C)高空间和/或时间分辨率的定量测量，以解决蛋白质相互作用的动力学问题。几种重要类型的蛋白质相互作用仍然很难用现有技术进行研究。例如，对膜蛋白相互作用(主要是乙二醇蛋白)的分析是具有挑战性的，因为这些蛋白在其天然的两亲性细胞环境之外不稳定。小分子(&lt；500 Da，包括绝大多数代谢物和药物)与蛋白质之间的相互作用动力学分析也缺乏，因为这些分子太小，不适合荧光标记，结合信号太弱，不适合无标记检测方法。同样有问题的是蛋白质翻译后修饰的特征，这种修饰由于翻译后附着一个小功能基团而改变了蛋白质的行为。具体地说，我们提出了一种电化学增强等离子体成像(ECEPI)系统来满足蛋白质相互作用动力学定量分析的关键需求，包括研究天然细胞状态下的膜蛋白质相互作用的能力，小分子相互作用和翻译后修饰的表征，用于亚细胞过程研究的高空间和时间分辨率的相互作用测量，以及以多细胞和微阵列格式执行高通量分析。ECEPI系统依赖于三项核心技术的精心集成：1)已被生物传感仪器公司(BI)成功商业化的电化学表面等离子共振系统(BI)，其独特的能力和坚实的性能，2)BI目前正在开发的用于高通量相互作用分析的专有高分辨率无失真棱镜表面等离子共振(SPR)成像系统，以及3)亚利桑那州立大学发明的高灵敏度阻抗成像技术。该项目的成功将导致一种新的工具，它能够：1) 蛋白质相互作用动力学的无标记实时识别和量化；2)蛋白质翻译后修饰的实时表征；3)小分子与蛋白质相互作用的定量测量；4)用基于细胞的分析原位量化膜蛋白(和糖蛋白)在其天然细胞环境中的相互作用；5)亚细胞过程的高分辨率分析；6)多细胞和微阵列形式的高通量分析

DESCRIPTION (provided by applicant): We are proposing a technology to help in three key areas of proteomics including (a) recognition of protein interactions, (b) characterization of post translational modifications, and (c) quantitative measurements at high spatial and/or temporal resolution to address the dynamics of protein interactions. Several significant types of protein interactions remain difficult to study with existing technologies. For example, the analysis of membrane protein interactions (mostly glycol proteins) is challenging, because these proteins are not stable outside of their native amphiphilic cellular environment. Analysis of interaction kinetics between small molecules (<500 Da, including a vast majority of metabolites and drugs) and proteins is also lacking, because these molecules are too small for fluorescence labeling, and the binding signals are too weak for label-free detection methods. Similarly problematic is the characterization of protein post-translational modifications, which alter protein behavior due to the attachment of a small functional group after translation. Specifically, we propose an electrochemically-enhanced plasmonic imaging (ECEPI) system to address key needs for quantitative analysis of protein interaction dynamics, including the ability to study membrane protein interactions in their native cellular state, characterization of small molecule interaction and post-translational modifications, measurement of interactions at high spatial and temporal resolution for the study of sub-cellular processes, and performing high-throughput analysis in multi-cellular and microarray formats. The ECEPI system relies upon careful integration of three core technologies: 1) the electrochemical surface plasmon resonance systems that have been successfully commercialized by Biosensing Instrument Inc. (BI) for their unique capabilities and solid performance, 2) a proprietary high resolution distortion-free prism-based surface plasmon resonance (SPR) imaging system currently under development at BI for high-throughput interaction analysis, and 3) a highly sensitive impedance imaging technique invented at Arizona State University. The success of this project will lead to a new instrument that is capable of: 1) Label-free real-time recognition and quantification of protein interaction kinetics; 2) Real-time characterization of post-translational modifications of proteins; 3) Quantitative measurement of small molecule interactions with proteins; 4) In situ quantification of membrane protein (and glycoprotein) interactions in their native cellular environment with cell-based assay; 5) High-resolution analysis of sub-cellular processes and; 6) High-throughput analysis in multi-cellular and microarray formats