Protein labeling in living cells - A combination of enzyme mediated peptide labeling and inverse electron demand Diels-Alder cycloaddition

我们最近开发了一种蛋白质标记策略，通过提供最小破坏性技术（最小标签尺寸），对活细胞中的选择性蛋白质标记产生重大影响。这种标记策略现在将作为一种新的蛋白质特异性化学蛋白质组学工具的关键技术，我们称之为ProSpecT（蛋白质特异性三功能捕获探针），用于识别受体-配体相互作用。该工具最有价值的资产是其在活细胞、组织或生物体中的潜在应用，这使得它在相互作用组学和蛋白质组学领域具有很高的吸引力。首先，我们将设计和合成不同变体的prospect探针，然后将在体外实验中基于一个众所周知的受体-配体对，即促红细胞生成素和人促红细胞生成素受体的细胞外结构域的可溶性形式进行评估。然后，在概念验证实验中，我们将展示我们可以从活细胞中捕获和识别已知的人类血管内皮生长因子（VEGF）受体。我们提案的最终目标是确定未知的细胞表面受体-配体相互作用，并证明ProSpecT是一种高度通用的试剂和方法，可作为功能基因组学筛选的新型关键方法。这项新技术将广泛应用于生物医学研究界，并对我们对生物途径和人类疾病机制的理解产生重大影响。

We recently developed a protein labeling strategy that makes significant impact with regard to selective protein labeling in live cells by providing a minimally disruptive technology (minimal tag size). This labeling strategy will now serve as key technology for a novel protein specific chemoproteomic tool which we call ProSpecT (protein specific trifunctional capture probe) for the identification of receptor-ligand interactions. Most valuable asset of the tool is its potential application in live cells, tissues or organisms which makes it highly attractive for the field of interactomics and proteomics. First, we will design and synthesize different variants of ProSpecT-probes which then will be evaluated in an in vitro assay based on a well-known receptor-ligand pair, namely erythropoietin and the soluble form of the extracellular domain of the human erythropoietin receptor. Then, in a proof of concept experiment we will show that we can capture and identify the known receptors of the human vascular endothelial growth factor (VEGF) from living cells. The ambitious final goal of our proposal is to identify unknown cell surface receptor-ligand interactions and to demonstrate that ProSpecT is a highly versatile reagent and method to serve as a novel key method for functional genomics screens. The new technology will be broadly useful to the biomedical research community and can make significant impact on our understanding of the mechanisms of biological pathways and human diseases.