HIGHLY PARALLEL EDMAN SEQUENCING OF INDIVIDUAL PEPTIDE MOLECULES

单个肽分子的高度并行 EDMAN 测序

• 批准号: 7944633
• 负责人: Robi D Mitra
• 金额: $ 23.14万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7944633
• 关键词: Algorithms; Amino Acid Sequence; Amino Acids; Antibodies; Arts; Binding; Biological Markers; Biological Process; Cancer cell line; Cell physiology; Cleaved cell; Conditioned Culture Media; Detection; Dipeptides; Disease; Dyes; Failure; Fluorescent Antibody Technique; Glass; Human; Image; Individual; Label; Lead; Malignant neoplasm of prostate; Maps; Measures; Methodology; Methods; N-terminal; Peptides; Phosphoserine; Phosphotyrosine; Protein Analysis; Proteins; Proteome; Proteomics; Protocols documentation; Sampling; Sensitivity and Specificity; Serum; Slide; Solutions; Surface; Technology; Time; Work; base; cost; digital; innovation; phenylisothiocyanate; prevent; protein aminoacid sequence; public health relevance; research study; single molecule; tool

DESCRIPTION (provided by applicant): Most biological processes are executed by proteins, but no method currently exists to accurately measure protein abundance and post-translational state proteome-wide. To redress this deficiency, we propose Digital Analysis of Proteins by End Sequencing (DAPES), a method that sequences many individual peptide molecules in parallel using Edman degradation. DAPES will be cost-effective, highly sensitive, and quantitative. DAPES is based on two innovations - 1) the use of dye-labeled antibodies to inexpensively and robustly detect single peptide molecules; and 2) a strategy that uses a universal set of ~20 antibodies to sequence peptide molecules. Our previous work, in which we used fluorescent antibodies to detect and quantify protein levels by single molecule counting, demonstrates that this approach is realistic and powerful. PUBLIC HEALTH RELEVANCE: Most cellular functions are performed by proteins, yet current methods are unable to accurately quantify protein levels and post-translational state in a comprehensive manner. This shortcoming is preventing a quantitative understanding of normal cellular processes, the mechanisms by which they fail, and how these failures lead to disease. To redress this deficiency, we propose to apply recent advances in single-molecule imaging to the field of protein detection. By sequencing single peptide molecules in parallel we will develop a protein analysis tool with unprecedented sensitivity, dynamic range, and utility.

描述（由申请人提供）：大多数生物过程是由蛋白质执行的，但目前不存在准确测量蛋白质丰度和整个蛋白质组翻译后状态的方法。为了弥补这一缺陷，我们提出了通过末端测序进行蛋白质数字分析 (DAPES)，这是一种使用 Edman 降解并行对许多单个肽分子进行测序的方法。 DAPES 将具有成本效益、高度灵敏且定量。 DAPES 基于两项创新 - 1) 使用染料标记的抗体来廉价且可靠地检测单个肽分子； 2) 使用约 20 种通用抗体对肽分子进行测序的策略。我们之前的工作使用荧光抗体通过单分子计数来检测和量化蛋白质水平，这表明这种方法是现实且强大的。 公共健康相关性：大多数细胞功能是由蛋白质执行的，但目前的方法无法以全面的方式准确量化蛋白质水平和翻译后状态。这一缺陷阻碍了对正常细胞过程、它们失败的机制以及这些失败如何导致疾病的定量理解。为了弥补这一缺陷，我们建议将单分子成像的最新进展应用于蛋白质检测领域。通过并行测序单个肽分子，我们将开发一种具有前所未有的灵敏度、动态范围和实用性的蛋白质分析工具。