HIGHLY PARALLEL EDMAN SEQUENCING OF INDIVIDUAL PEPTIDE MOLECULES

描述（由申请人提供）：大多数生物过程是由蛋白质执行的，但目前还没有方法可以精确测量蛋白质丰度和蛋白质组翻译后状态。为了纠正这一缺陷，我们提出了通过末端测序的蛋白质数字分析（DAPES），这是一种利用Edman降解平行测序许多单个肽分子的方法。dape将具有成本效益，高度敏感和定量。DAPES基于两项创新：1)使用染料标记的抗体来廉价而可靠地检测单个肽分子；2)使用一组通用的约20种抗体对肽分子进行测序的策略。在我们之前的工作中，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，这表明这种方法是现实的和强大的。

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.