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利用发现技术绘制蛋白质相互作用组图谱， 与当前最先进的技术相比，时空分辨率提高了几个数量级。这 这项工作的动机是推动蛋白质相互作用组发现技术的分辨率超越关键 里程碑，例如单细胞和单一蛋白质阈值，将产生类似于类似的领域范围的影响 转录组学和显微镜学的进展。我们的统一方法由 R35 MIRA 奖项资助， 结合了光催化邻近标记，其中光动力催化剂连接到亲和手柄驱动器上 合成亲和探针与附近蛋白质的交联，具有图案光和相互作用门控 激活以同时实施多个维度的特异性。在实验室工作的第一年 重点关注我们提出的两个目标：（1）高通量发现内源肽结合靶标，以及 (2)单细胞解析空间相互作用组学。内源肽代表蛋白质的主要部分 人类细胞中的物质，但驱动其生物功能的机制实际上仍未被探索。一 我们工作的主旨将光催化邻近标记与快速离子迁移蛋白质组学相结合，以建立其 受体，绘制肽组和蛋白质组之间的界面。这项工作的关键是高效 催化剂-肽缀合物的合成。我们演示了多达十种缀合物的制备和分离 使用质量定向制备 HPLC 在 3 小时内完成，每年可支持多达 800 个目标 ID 实验，以及 通过光催化标记和蛋白质组学证明了肽目标 ID。我们的工作是开发空间 相互作用组学技术也取得了长足的进步。我们已经构建了定制的硬件、软件和 试剂并用它们来演示机器视觉引导的图案照射和光催化蛋白质 以单细胞分辨率标记扁桃体中的 CD3+ 细胞。这些项目以及 R35 范围内的未来工作 MIRA 奖涉及重要的常规和高通量有机合成和纯化，其中包括许多 中间体和最终产品需要制备型质谱定向 HPLC。在最初的 R35 MIRA 应用中 我们提议使用制备型 HPLC-MS（Waters AutoPurification System 2000 系列）由邻近的实验室拥有。然而，该组（Tri-I TDI）是 从威尔康奈尔医学院转到洛克菲勒大学，该仪器将不再可用。 由于他们离开该机构的意外性质以及缺乏任何同等的文书 威尔康奈尔医学中心的核心设施或 PI 领导的实验室，我们正在请求补充资金来购买 更换仪器（新的沃特世自动净化系统），成本不到 250,000 美元。我们是 请求资助仪器和必要配件的全部费用，请表明当前资助 Geri 实验室的支持不足以在没有行政补充的情况下购买仪器，并且 有足够的资金来支持、维护和操作要购买的仪器。