Electrochemically-Enhanced Plasmonic Imaging for Quantitative Proteomics

用于定量蛋白质组学的电化学增强等离子体成像

• 批准号: 8661191
• 负责人: Nguyen Ly
• 金额: $ 17.67万
• 依托单位: BIOSENSING INSTRUMENT, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-15 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8661191
• 关键词: Address; Algorithms; Antibodies; Applications Grants; Area; Arizona; Behavior; Binding; Biological Assay; Biological Models; Biosensing Techniques; Biosensor; Carbonic Anhydrase II; Cell Surface Proteins; Cell physiology; Cells; Cultured Cells; Data Collection; Detection; Development; Drug Interactions; Environment; Fluorescence; Glycols; Glycoproteins; Health; Image; Imaging Techniques; Imaging technology; In Situ; Institutes; Kinetics; Label; Lead; Measurement; Membrane Glycoproteins; Membrane Proteins; Methods; Monitor; Peptides; Performance; Pharmaceutical Preparations; Phase; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Printing; Protein Analysis; Protein Binding; Protein Dynamics; Protein Microchips; Proteins; Proteomics; Reporting; Research; Resolution; Sampling; Signal Transduction; Small Business Innovation Research Grant; Solid; Surface Plasmon Resonance; System; Technology; Testing; Time; Translations; United States National Institutes of Health; Universities; base; density; electric impedance; functional group; high throughput analysis; inhibitor/antagonist; instrument; new technology; plasmonics; protein function; protein protein interaction; prototype; response; small molecule; software development; success

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

描述（由申请人提供）：我们提出了一种技术，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）表征后， 翻译修饰，和（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。几种重要类型的蛋白质相互作用仍然难以用现有技术进行研究。例如，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，因为这些蛋白质在其天然两亲性细胞环境之外是不稳定的。小分子（<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，因为这些分子太小而无法进行荧光标记，并且结合信号对于无标记检测方法来说太弱。类似的问题是蛋白质翻译后修饰的表征，其由于翻译后小官能团的附着而改变蛋白质行为。具体而言，我们提出了一种电化学增强等离子体成像（ECEPI）系统，以解决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膜蛋白相互作用的能力，小分子相互作用和翻译后修饰的表征，以高空间和时间分辨率测量亚细胞过程研究的相互作用，以及以多细胞和微阵列形式进行高通量分析。ECEPI系统依赖于三个核心技术的精心集成：1）电化学表面等离子体共振系统，已被Biosensing Instrument Inc.成功商业化。(BI)凭借其独特的功能和可靠的性能，2）BI目前正在开发的专有高分辨率、无失真、基于棱镜的表面等离子体共振（SPR）成像系统，用于高通量相互作用分析，以及3）亚利桑那州发明的高灵敏度阻抗成像技术州立大学。该项目的成功将导致一种新的仪器，能够：1） 蛋白质相互作用动力学的无标记实时识别和定量; 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）膜蛋白的原位定量（和糖蛋白）在它们的天然细胞环境中的相互作用与基于细胞的测定; 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