HIGHLY PARALLEL EDMAN SEQUENCING OF INDIVIDUAL PEPTIDE MOLECULES

描述（由申请人提供）：大多数生物过程由蛋白质执行，但目前还没有方法可以准确测量蛋白质丰度和蛋白质组范围内的翻译后状态。为了弥补这一不足，我们提出了末端测序（DAPES）的蛋白质数字分析，这是一种使用Edman降解并行测序许多单个肽分子的方法。DAPES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和稳健地检测单个肽分子; 2）使用通用的~20种抗体来测序肽分子的策略。我们以前的工作，其中我们使用荧光抗体来检测和定量蛋白质水平的单分子计数，表明这种方法是现实的和强大的。 公共卫生相关性：大多数细胞功能由蛋白质执行，但目前的方法无法以全面的方式准确地定量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的定量理解，它们失败的机制以及这些失败如何导致疾病。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。通过对单个肽分子进行平行测序，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.