Vibrational spectroscopy with metasurface optics (VISMO) for high-throughput identification of protein post-translational modifications

项目摘要/摘要 蛋白质对广泛的细胞过程至关重要--从细胞信号到免疫反应，再到营养 运输、生长和代谢调节。蛋白质的正常功能敏感地依赖于类型， 位置和大量的翻译后修饰(PTM)。PTM属性中的像差可以 降低蛋白质的功能，损害它们所在细胞的生理和功能。的确， 突变的PTM与多种疾病有关，包括癌症、自身免疫性疾病、 神经退行性疾病和炎症。核酸测序几乎无法洞察PTM 属性，以及敏感和高通量的PTM的定性和量化仍然令人望而生畏 质谱学(MS)和免疫分析的挑战。南瓜种子公司正在开发一种基于光学和 一种无标记法测量蛋白质PTM的方法，称为超表面光学振动光谱 (Vismo)。Vismo提供对数百万种蛋白质的高速分析--包括它们的序列、结构和 相互作用-与其他蛋白质组解决方案相比具有三个关键优势：(1)在无标签格式中，我们 通过振动散射光谱唯一地识别每个分子，而不是依赖较低的分辨率 荧光标记。(2)使用纳米结构的硅芯片，我们制造了高效的光学谐振器，既 显著放大信噪比--即使对于丰度很低的生物标志物也是如此--并增加样本 吞吐量，同时测量每平方厘米约300万个蛋白质。(3)我们使用 尖端机器学习(ML)算法，从振动光谱中剖析信息丰富的特征， 包括与蛋白质的一级、二级和三级结构相对应的波数，如 以及与蛋白质或小分子结合有关的变化。我们的第一阶段提案将发展这一点 用于敏感、高通量和高分辨率蛋白质检测的革命性平台，包括他们的 丰度、序列和PTMS。AIM 1开发的传感器能够实现敏感的、从少数分子到单分子 光谱学，建立我们的特定氨基酸、短肽序列和PTMS的光谱目录； 并开发了机器学习(ML)算法，为每个分子的光学性质提供了可解释性 散射特征。目的2提出了我们的利用化学切割进行蛋白质测序的方法和我们的 获得专利的“减法测序”方法。第一阶段的结果将成为我们第二阶段工作的基础 建议，对整个蛋白质组测序和蛋白质图谱具有潜在的深远影响 细胞类型的曲目。生物治疗公司和学术研究人员研究完整的天然蛋白质 将重视南瓜种子公司的平台与现有的ELISA型检测相比提供的高分辨率，以及 与传统的质谱学相比，具有更高的灵敏度和吞吐量。我们的方法提供了一种新的 探索蛋白质结构和相互作用以揭示疾病的新维度的方法学 病因学和新治疗发展的独特机会。

Project Summary/Abstract Proteins are critical to a wide array of cellular processes - from cell signaling to immune responses, nutrient transport, growth, and metabolic regulation. Proper function of proteins depends sensitively on the type, location, and abundance of post-translational modifications (PTMs). Aberrations in PTM properties can degrade protein function and compromise the physiology and function of the cells in which they reside. Indeed, altered PTMs have been implicated in a variety of diseases, including cancers, auto-immune diseases, neurodegenerative diseases, and inflammation. Nucleic acid sequencing gives almost no insight into PTM attributes, and sensitive and high-throughput characterization and quantification of PTMs remains a daunting challenge for mass spectroscopy (MS) and immunoassays. Pumpkinseed is developing an optics-based and label-free approach to measure protein PTMs, termed Vibrational Spectroscopy with Metasurface Optics (VISMO). VISMO offers high-speed analysis of millions of proteins – including their sequence, structure, and interactions – with three key advantages over other proteomics solutions: (1) In a label-free format, we uniquely identify each molecule via its vibrational scattering spectra, rather than relying on lower-resolution fluorescent tagging. (2) Using nanostructured Si chips, we create highly efficient optical resonators that both dramatically amplify the signal-to-noise ratio – even for very low abundance biomarkers – and increase sample throughput, with simultaneous measurement of ~3 million proteins per square centimeter. (3) We use cutting-edge machine learning (ML) algorithms to dissect information-rich features from the vibrational spectra, including the wavenumbers that correspond to the primary, secondary, and tertiary structure of the protein, as well as changes related to protein or small-molecule binding. Our Phase I proposal will develop this revolutionary platform for sensitive, high-throughput, and high-resolution protein detection, including their abundance, sequence, and PTMs. Aim 1 develops sensors that enable sensitive, few-to-single-molecule spectroscopy, builds our catalog of spectra for particular amino acids, short peptide sequences, and PTMs; and develops machine learning (ML) algorithms that provide interpretability to each molecule’s optical scattering signature. Aim 2 advances our methods for protein sequencing using chemical cleavage and our patented ‘sequencing by subtraction’ methodology. Phase I results will form the foundation of our Phase II proposal, with potentially far-reaching impacts on whole-proteome sequencing and protein-profiling of the vast repertoire of cell types. Biotherapeutic companies and academic researchers studying intact, native proteins will value the high-resolution that Pumpkinseed’s platform offers compared to existing ELISA-type assays, and the higher-sensitivity and throughput compared to traditional mass spectrometry. Our approach provides a new methodology for probing protein structure and interactions in order to reveal new dimensions of disease etiology and distinct opportunities for new therapeutic development.