Supplement for Optically Gated Discovery of Protein-Biomolecule Interactions project.

蛋白质-生物分子相互作用光门发现项目的补充。

• 批准号: 10807688
• 负责人: Jacob Geri
• 金额: $ 23.8万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-24 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10807688
• 关键词: Administrative Supplement; Affinity; Award; Binding; Biological Process; CD3 Antigens; Cells; Computer software; Core Facility; Data; Development; Dimensions; Disease; Funding; Future; Goals; Grant; Health; High Pressure Liquid Chromatography; Hour; Human; Institution; Label; Laboratories; Light; Maps; Medicine; Microscopy; Motivation; Nature; Optics; Organic Synthesis; Pattern; Peptides; Preparation; Proteins; Proteome; Proteomics; Reagent; Research; Resolution; Series; Specificity; Synthesis Chemistry; System; Technology; Tonsil; Universities; Water; Work; catalyst; cost; crosslink; diagnostic strategy; equipment acquisition; experimental study; improved; instrument; ion mobility; irradiation; machine vision; new technology; novel diagnostics; novel therapeutics; receptor; spatiotemporal; transcriptomics; virtual

The overall goal of research in the Geri lab is to map protein interactomes using discovery technologies that provide orders of magnitude improvements in spatiotemporal resolution over the current state-of-the-art. The motivation for this work is that advancing the resolution of protein interactome discovery technology beyond key milestones, such as single cell and single protein thresholds, will have a field-wide impact analogous to similar advances in transcriptomics and microscopy. Our unified approach, funded through a R35 MIRA award, combines photocatalytic proximity labeling, in which light-powered catalysts attached to an affinity handle drive the crosslinking of synthetic affinity probes with nearby proteins, with patterned light and interaction-gated activation to simultaneously enforce multiple dimensions of specificity. The first year of work in the lab has focused on two of our proposed aims: (1) high throughput discovery of endogenous peptide binding targets, and (2) single-cell resolved spatial interactomics. Endogenous peptides represent a major fraction of proteinaceous matter in human cells, but the mechanisms powering their biological functions remain virtually unexplored. One thrust of our work merges photocatalytic proximity labeling with rapid ion-mobility proteomics to establish their receptors, mapping the interface between the peptidome and the proteome. Key to this effort is the efficient synthesis of catalyst-peptide conjugates. We demonstrated the preparation and isolation of up to ten conjugates in 3 hours using mass directed prep HPLC, which can support up to 800 target ID experiments per year, and demonstrated peptide target ID through photocatalytic labeling and proteomics. Our work to develop spatial interactomics technology has also made substantial progress. We have built bespoke hardware, software, and reagents and used these to demonstrate machine vision-guided patterned irradiation and photocatalytic protein labeling of CD3+ cells in tonsil at single-cell resolution. These projects, and future work in the scope of the R35 MIRA award, entails significant of routine and high throughput organic synthesis and purification, with many intermediates and final products requiring preparatory mass directed HPLC. In the original R35 MIRA application we proposed to use, and during the first year of award activities we have used, a prep HPLC-MS (Waters AutoPurification System 2000 Series) owned by a neighboring laboratory. However, this group (Tri-I TDI) is moving from Weill Cornell Medicine to Rockefeller University, and this instrument will no longer be available. Due to the unexpected nature of their departure from the institution and the lack of any equivalent instrument in a core facility or PI-led laboratory at Weill Cornell Medicine, we are requesting supplemental funding to purchase a replacement instrument (a new Waters AutoPurification System), which costs less than $250,000. We are requesting funding for the entire cost of the instrument and necessary accessories, show that current grant support of the Geri lab is insufficient to purchase the instrument without an administrative supplement, and that sufficient funding is available to support, maintain, and operate the instrument to be purchased.

GERI实验室研究的总体目标是使用发现技术来绘制蛋白质相互作用， 提供比当前最新分辨率的时空分辨率的数量级改善。这 这项工作的动机是，将蛋白质Interactome互动发现技术的分辨率超越关键 里程碑，例如单细胞和单蛋白阈值，将具有类似于类似的野外影响 转录组学和显微镜的进展。我们的统一方法，通过R35 MIRA奖资助， 结合光催化接近标记，其中连接到亲和力手柄驱动器的轻催化剂 合成亲和力探针与附近蛋白质的交联，带有图案光和相互作用门控 激活同时执行特异性的多个维度。实验室工作的第一年 专注于我们提出的两个目标：（1）高吞吐量发现内源性肽结合靶标，并 （2）单细胞分辨的空间相互作用组学。内源性肽代表蛋白质的主要部分 人类细胞中的物质，但是为其生物学功能提供动力的机制几乎没有探索。一 我们作品的推力将光催化接近标记与快速离子动力蛋白质组学合并以建立其 受体，映射肽组和蛋白质组之间的界面。这项努力的关键是有效的 催化剂肽结合物的合成。我们证明了多达十个共轭的准备和隔离 使用质量定向PREP HPLC在3小时内，每年最多可以支持800个目标ID实验，并且 通过光催化标记和蛋白质组学证明了肽靶ID。我们发展空间的工作 Interactomics技术也取得了长足的进步。我们已经建立了定制的硬件，软件和 试剂并用它们证明机器视觉引导的图案辐射和光催化蛋白 单细胞分辨率在扁桃体中标记CD3+细胞的标记。这些项目以及R35范围的未来工作 MIRA奖具有重要的常规和高吞吐量有机合成和纯化，许多 中间产品和最终产品需要预先批量的HPLC。在原始R35 MIRA应用中 我们建议使用，在我们使用的奖励活动的第一年中 自动化系统2000系列）由附近的实验室拥有。但是，这个组（tri-i tdi）是 从威尔·康奈尔（Weill Cornell）医学到洛克菲勒大学（Rockefeller University），该乐器将不再可用。 由于他们离开机构的意外性质以及缺乏同等工具 Weill Cornell Medicine是一个核心设施或PI-LED实验室，我们要求补充资金购买 一种替代工具（一种新的水域自动化系统），价格低于25万美元。我们是 要求为仪器和必要配件的全部成本提供资金，表明当前赠款 GERI实验室的支持不足以在没有管理补充的情况下购买该工具，并且 有足够的资金用于支持，维护和操作要购买的工具。