Peptide detection using the proximity ligation assay

使用邻近连接测定进行肽检测

• 批准号: 6835030
• 负责人: Jay R Hesselberth
• 金额: $ 4.3万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6835030
• 关键词: bioassay; biotechnology; diagnosis design /evaluation; diagnosis quality /standard; disease /disorder; microarray technology; nucleic acid sequence; oligonucleotides; polymerase chain reaction; postdoctoral investigator; posttranslational modifications; protein quantitation /detection; protein sequence; proteomics

DESCRIPTION (provided by applicant): Proteomic analyses are revolutionizing the diagnosis of human disease. Existing technologies are able to pinpoint the proteins indicative of disease progression, enabling their use in early detection and treatment scenarios. However, the use of proteomic techniques is limited because of the complexities of their usage and data interpretation. As an alternative to the current methods of peptide and protein analysis, I propose employing novel peptide-specific nucleic acid aptamers in the Proximity Ligation Assay for the detection of unique peptides in complex mixtures. In this system, a pair of aptamers would recognize the terminal ends of a peptide and, by virtue of their proximity, be enzymatically ligated together. Because most peptides are uniquely specified by their terminal sequences, the ligation product indicates a unique peptide. The presence of a peptide can be assessed via a PCR reaction, instead of its identification by mass. Using this novel method, one could quantitate peptide concentrations, identify low-abundance peptides and detect post-translation modifications, potentially in a single reaction. We envision adapting the method to readout via a microarray, simplifying complex protein mixture measurements to a single chip platform.

描述（由申请人提供）： 蛋白质组学分析正在彻底改变人类疾病的诊断。现有技术能够精确定位指示疾病进展的蛋白质，使其能够用于早期检测和治疗方案。然而，由于其使用和数据解释的复杂性，蛋白质组学技术的使用受到限制。作为替代目前的方法的肽和蛋白质分析，我建议采用新的肽特异性核酸适体的邻近连接试验检测复杂的混合物中的独特的肽。在该系统中，一对适体将识别肽的末端，并且凭借它们的接近性，被酶促连接在一起。因为大多数肽由其末端序列唯一指定，所以连接产物指示唯一肽。肽的存在可以通过PCR反应评估，而不是通过质量鉴定。使用这种新方法，人们可以定量肽浓度，鉴定低丰度肽和检测翻译后修饰，可能在一个单一的反应。我们设想通过微阵列将该方法调整为读出，将复杂的蛋白质混合物测量简化为单个芯片平台。