Single-molecule protein sequencing by detection and identification of N-terminal amino acids

通过检测和鉴定 N 端氨基酸进行单分子蛋白质测序

基本信息

批准号：
10646060
负责人：
Daniel Masao Estandian
金额：
$ 39.06万
依托单位：
GLYPHIC BIOTECHNOLOGIES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-15 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10646060
关键词：
Acceleration Affinity Amino Acid Sequence Amino Acids Antibodies Antibody Affinity Area Basic Science Binding Biological Biological Assay Biological Markers Biology Biotechnology Blood Charge Chemicals Chemistry Complex Complex Mixtures DNA sequencing Detection Development Diagnostics Research Disease Dyes Enzyme-Linked Immunosorbent Assay Face Fluorescence Microscopy Fluorescence Resonance Energy Transfer Functional disorder Future Genomics Growth and Development function Hour Image Immune Sera Immunity Immunize Individual Infection Knowledge Label Libraries Ligation Llama Malignant Neoplasms Marketing Mass Spectrum Analysis Measures Methods Monoclonal Antibodies Mus N-terminal Noise Oryctolagus cuniculus Parents Patients Peptide Mapping Peptide Sequence Determination Peptides Phase Process Protein Analysis Proteins Proteome Proteomics Reaction Reagent Research Resolution Sampling Sensitivity and Specificity Side Signal Transduction Solid Specificity Surface Surface Plasmon Resonance System Technology Tertiary Protein Structure Testing Titrations Tumor Antigens Visualization Yeasts antibody engineering clinical diagnosis clinical diagnostics commercialization cost effective cross reactivity digital disease diagnosis immunological diversity improved innovation manufacture molecular mass nanopore next generation next generation sequencing novel novel strategies pathogen polyclonal antibody protein complex protein expression protein function protein structure sequencing platform single molecule success tumor

项目摘要

SUMMARY - Subtle changes in protein expression are critical for proper growth and development, but irregu- larities can cause deleterious cellular effects or large-scale biological dysfunction. Sequencing samples with complex mixtures of proteins could greatly accelerate research into protein function and biology, but there is currently no efficient and cost-effective strategy for protein sequencing at single-amino-acid resolution. Two methods are commercially available for protein sequencing. In the first, “Edman degradation”, bulk quanti- ties of whole protein or purified fragments are sequenced by cleaving the first (N-terminal) amino acid and chem- ically identifying it. In the second method, based on mass spectrometry, a single protein or mixture of proteins is fragmented, and the molecular mass and charge of each fragment are analyzed. This information is compared known protein sequences to infer the identity of the input proteins. Both of these methods require ~1 million molecules of each protein, and Edman degradation cannot currently be used on heterogenous protein mixtures. Existing approaches for single molecule protein sequencing are hindered by the number and diversity of amino acids, as well as the interactions between amino acids that interfere with chemical identification of their side chains. Harsh denaturation agents can mitigate some issues, but they can compromise the identification systems themselves. In addition, denaturation agents only remove some of the intramolecular interactions of proteins. Glyphic Biotechnologies is developing a novel strategy to sequence individual protein molecules in their entirety from a heterogeneous sample. This process is based on ligating the N-terminal amino acid to a cleavable chem- ical linker, which subsequently tethers it locally to the surface. Cleavage of the linker removes the N-terminal amino acid from the protein for highly sensitive identification with no interference from protein structure or adja- cent amino acids. The process is repeated for each subsequent amino acid, yielding the protein sequence. The approach may simultaneously sequence millions to billions of individual protein molecules in hours, which will revolutionize protein analysis by making large-scale protein sequencing feasible, inexpensive, and routine. The current proposal focuses on developing reagents specifically to detect the N-terminal amino acid of proteins, allowing amino acids to be digitally identified via this N-terminal isolation strategy. In Aim 1 we will generate antibodies to recognize at least 10 different isolated amino acids – enough to identify ~90% of the proteome after 10 sequencing rounds. In Aim 2 we will further optimize the antibodies and demonstrate the feasibility of using them to sequence individual proteins among a background of non-modified proteins. Success of these Aims will enable the Glyphic protein sequencing platform to detect, quantify, and sequence single proteins in complex protein mixtures in an unbiased fashion - without any prior knowledge of their identity or even their existence. When commercialized, it will enable clinical diagnosis of disease based on the proteins present in a patient sample and allow identification of unique proteins to for as-yet unknown biomarkers.

摘要 - 蛋白质表达的细微变化对于适当的生长和发育至关重要，但是纤维可能会导致删除的细胞效应或大规模生物功能障碍。与样品进行测序蛋白质的复杂混合可能会大大加速蛋白质功能和生物学的研究，但是有目前，在单氨基酸分辨率下尚无蛋白质测序的有效且具有成本效益的策略。两种方法可用于蛋白质测序。首先，“埃德曼退化”，大量量化通过裂解第一（N末端）氨基酸和化学来对全蛋白或纯化片段的关系进行测序 ifully识别它。在第二种方法中，基于质谱法，蛋白质的单蛋白或混合物是分散，分析每个片段的分子质量和电荷。比较此信息已知的蛋白质序列来推断输入蛋白的身份。这两种方法都需要约100万每种蛋白质的分子和Edman降解目前不能用于异质蛋白混合物。单分子蛋白测序的现有方法受到氨基的数量和多样性的阻碍酸以及干扰其侧化学鉴定的氨基酸之间的相互作用链。苛刻的变性代理可以减轻某些问题，但它们可以损害身份系统自己。另外，变性剂仅去除蛋白质的某些分子内相互作用。甘氨酸生物技术正在制定一种新的策略，以整体对单个蛋白质分子进行测序来自异质样品。该过程基于将N末端氨基酸连接到可裂解的化学 iCal接头，随后将其局部置于表面。链接器的切割消除了N末端来自蛋白质的氨基酸，用于高度敏感的鉴定，而不会受到蛋白质结构或相邻的干扰百分钟氨基酸。为每个随后的氨基酸重复该过程，从而产生蛋白质序列。方法可以简单地将数百万到数十亿个单独的蛋白质分子在小时内序列，这将通过使大规模的蛋白质测序可行，廉价和常规来彻底改变蛋白质分析。当前的建议着重于开发专门检测蛋白质N末端氨基酸的试剂，即通过这种N末端分离策略，可以通过数字识别氨基酸。在AIM 1中，我们将产生识别至少10种不同分离的氨基酸的抗体 - 足以鉴定约90％的蛋白质组 10个测序回合。在AIM 2中，我们将进一步优化抗体，并证明使用的可行性它们以在非修饰蛋白的背景之间对单个蛋白质进行测序。这些目标的成功将使甘氨酸蛋白测序平台能够检测，量化和序列复杂蛋白质中的单蛋白以公正的方式混合 - 没有任何先验知识甚至它们的存在。商业化时，它将基于蛋白质对疾病进行临床诊断存在于患者样品中，并允许鉴定独特的蛋白质，以使其尚未知道的生物标志物。