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正常生长和发育至关重要，但不规则惰性气体可能会导致有害的细胞效应或大规模的生物功能障碍。蛋白质的复杂混合物可以极大地加速蛋白质功能和生物学的研究，但是目前还没有有效且经济高效的单氨基酸分辨率蛋白质测序策略。有两种商业化的蛋白质测序方法，第一种是“Edman 降解”，批量定量。通过切割第一个（N 端）氨基酸和化学方法对整个蛋白质或纯化片段的连接进行测序。在第二种方法中，基于质谱法，单一蛋白质或蛋白质混合物被鉴定。片段化，并分析每个片段的分子质量和电荷。已知的蛋白质序列来推断输入蛋白质的身份这两种方法都需要约 100 万个。每种蛋白质的分子，Edman 降解目前不能用于异源蛋白质混合物。现有的单分子蛋白质测序方法受到氨基酸数量和多样性的阻碍酸，以及干扰其侧面化学鉴定的氨基酸之间的相互作用严酷的变性剂可以缓解一些问题，但它们可能会损害识别系统。此外，变性剂本身仅消除蛋白质的一些分子内相互作用。 Glyphic Biotechnologies 正在开发一种新策略，对单个蛋白质分子进行整体测序该过程基于将 N 末端氨基酸连接到可裂解的化学物质上。 ical 连接子，随后将其局部固定到表面。连接子的裂解会去除 N 末端。蛋白质中的氨基酸可进行高度灵敏的鉴定，不受蛋白质结构或邻接的干扰对每个后续氨基酸重复该过程，产生蛋白质序列。方法可以在数小时内同时对数百万至数十亿个单独的蛋白质分子进行测序，这将通过使大规模蛋白质测序变得可行、廉价且常规化，彻底改变蛋白质分析。目前的提案重点是开发专门检测蛋白质 N 端氨基酸的试剂，允许通过这种 N 端分离策略对氨基酸进行数字识别。在目标 1 中，我们将生成。识别至少 10 种不同的分离氨基酸的抗体 – 足以识别约 90% 的蛋白质组 10轮测序，在目标2中我们将进一步优化抗体并论证使用的可行性。他们对非修饰蛋白质背景中的单个蛋白质进行测序。这些目标的成功将使 Glyphic 蛋白质测序平台能够检测、定量和测序以公正的方式分析复杂蛋白质混合物中的单一蛋白质 - 无需事先了解其身份甚至当它们商业化时，它将能够根据蛋白质进行疾病的临床诊断。存在于患者样本中，并允许识别独特的蛋白质以用于迄今未知的生物标志物。