Proteome Capture in Hydrogel Beads for High Resolution Single Cell Analysis

水凝胶珠中的蛋白质组捕获用于高分辨率单细胞分析

• 批准号: 10483791
• 负责人: Peter Krutzik
• 金额: $ 25.06万
• 依托单位: CYTORUM, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-04 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10483791
• 关键词: Acrylamides; Antibodies; Antibody Affinity; Antibody Specificity; Antigen-Antibody Complex; Antigens; Biochemical; Biological Assay; Blood; Cell model; Cells; Cellular biology; Chemistry; Complex; Complex Mixtures; Cross-Linking Reagents; Crosslinker; Custom; Cytolysis; Detection; Development; Diagnostic; Disease; Environment; Epitopes; Exhibits; Flow Cytometry; Fluorescence; Gel; Grant; Growth; Heating; Hour; Hydrogels; Imaging Techniques; Immune response; Immunooncology; Label; Measurement; Measures; Membrane; Membrane Proteins; Methods; Microfluidics; Monitor; Nature; Noise; Optics; Pathway interactions; Peptides; Pharmaceutical Preparations; Phase; Population; Preparation; Procedures; Property; Protein Analysis; Protein Denaturation; Proteins; Proteome; Protocols documentation; Reagent; Resolution; STAT1 gene; Sampling; Signal Pathway; Signal Transduction; Small Business Innovation Research Grant; Speed; Stains; Surface; Surrogate Markers; System; Techniques; Technology; Temperature; Testing; Time; Tissues; Western Blotting; Work; antibody detection; antigen detection; crosslink; design; detection sensitivity; immune system function; improved; innovation; insight; instrument; instrumentation; light scattering; new technology; next generation; polyacrylamide; polyacrylamide hydrogels; polymerization; preservation; response; single cell analysis; tool

Summary Analysis of surface and intracellular proteins at the single cell level has become widespread in immunology, oncology, and diagnostics. Although several platforms have emerged for single cell analysis, flow cytometry has remained one of the most powerful due to its speed, sensitivity, and quantitative nature. With current instrumentation, 20-40 proteins can be measured per cell, at rates of thousands of cells per second. However, a major limitation of the platform is the number of antibody reagents that have been developed and validated for flow cytometry. Currently, less than 2% of intracellular protein targets can be measured by flow cytometry, while other biochemical techniques such as Western blotting are able to measure over 25%. This limitation in flow cytometry is largely due to the fact that the cells need to remain intact for analysis. This precludes sample preparation methods that would fully denature the proteins and expose the epitopes necessary for detection by a wider range of antibodies. Conversely, in Western blotting, the proteins are denatured and spread out on a membrane surface, making them accessible to antibodies which have been generated against linearized epitopes. Here we propose a technology platform that transfers the proteome of a single cell into a hydrogel bead. This enables the proteins to be denatured fully with harsh denaturation treatments, while also spreading them out in space to allow for easier access by antibody reagents, as the beads are larger than the original cells. The system is designed to preserve the critical advantages of single cell analysis by flow cytometry, such as the ability to stain for surface markers in complex immune cell populations, speed of acquisition, and detection sensitivity. The beads are also uniform in size, stable in solution, and optically clear, creating an ideal substrate for highly sensitive detection. By emulating the environment typically encountered in Western blots, we anticipate that the single cell bead platform will enable the use of hundreds to thousands of additional antibody reagents. This vast expansion in available reagents will enable next generation single cell analysis, the direct analysis of proteins instead of downstream or upstream surrogate markers, an increase in the number of signaling pathways that can be monitored, and ultimately deeper insight into disease states and drug activity.

总结 在单细胞水平上对表面和细胞内蛋白质的分析已经变得广泛， 免疫学肿瘤学和诊断学尽管已经出现了几种用于单细胞分析的平台， 流式细胞术由于其速度、灵敏度和定量性质而一直是最强大的方法之一。 利用目前的仪器，每个细胞可以测量20-40种蛋白质，以每个细胞数千个细胞的速率， 一下然而，该平台的一个主要局限性是已经使用的抗体试剂的数量。 开发并验证了流式细胞术。目前，只有不到2%的细胞内蛋白质靶点可以被靶向。 通过流式细胞术测量，而其他生物化学技术如Western印迹能够 超过25%。流式细胞术中的这种限制主要是由于细胞需要保留在细胞中。 完整地进行分析。这排除了将使蛋白质完全变性的样品制备方法， 暴露出通过更广泛的抗体进行检测所必需的表位。相反，在西方 蛋白质变性并分散在膜表面，使其易于被膜吸收。 针对线性化表位产生的抗体。在这里，我们提出了一个技术平台， 将单个细胞的蛋白质组转移到水凝胶珠中。这使得蛋白质能够变性 充分与苛刻的变性处理，同时也分散在太空中，让他们更容易获得 抗体试剂，因为珠子比原始细胞大。该系统旨在保护 通过流式细胞术进行单细胞分析的关键优势，例如染色表面的能力， 复杂免疫细胞群体中的标记物、采集速度和检测灵敏度。将珠粒 尺寸均匀，在溶液中稳定，光学透明，为高灵敏度的 侦测通过模拟蛋白质印迹中通常遇到的环境，我们预计， 单细胞珠平台将使得能够使用数百至数千种另外的抗体试剂。这 可用试剂的大量扩增将使下一代单细胞分析成为可能， 蛋白质而不是下游或上游替代标记物，信号转导的数量增加， 可以监测的途径，并最终更深入地了解疾病状态和药物活性。