HIGHLY PARALLEL EDMAN SEQUENCING OF INDIVIDUAL PEPTIDE MOLECULES

描述(申请人提供)：大多数生物过程是由蛋白质执行的，但目前还不存在准确测量蛋白质组范围内蛋白质丰度和翻译后状态的方法。为了弥补这一不足，我们提出了蛋白质末端测序的数字分析(DAPES)，这是一种使用埃德曼降解来并行对许多单独的多肽分子进行测序的方法。DAPE将是经济高效、高度敏感和量化的。DAPES基于两项创新-1)使用染料标记的抗体来廉价而有力地检测单肽分子；2)使用一套通用的~20种抗体对多肽分子进行测序。我们之前的工作，我们使用荧光抗体通过单分子计数来检测和量化蛋白质水平，证明了这种方法是现实的和强大的。 公共卫生相关性：大多数细胞功能是由蛋白质执行的，但目前的方法无法全面准确地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、失败机制以及这些失败如何导致疾病的定量了解。为了弥补这一不足，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。通过对单肽分子进行并行测序，我们将开发出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。

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.