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

• 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
• 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

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-蛋白质表达的微妙变化对正常生长和发育至关重要，但不规则的蛋白质表达对正常生长和发育至关重要。 空隙可引起有害的细胞效应或大规模的生物功能障碍。测序样品， 蛋白质的复杂混合物可以大大加速对蛋白质功能和生物学的研究， 目前还没有有效的和成本有效的策略用于以单个氨基酸分辨率进行蛋白质测序。 有两种方法可用于蛋白质测序。在第一，“埃德曼降解”，散装定量- 通过切割第一个（N-末端）氨基酸和化学标记来对整个蛋白质或纯化片段的序列进行测序。 在第二种方法中，基于质谱法， 片段化，并分析每个片段的分子量和电荷。这些信息是比较 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~ 100万 每种蛋白质的分子，并且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序列的数量和多样性的阻碍， 酸，以及氨基酸之间的相互作用，干扰其侧的化学鉴定 店苛刻的变性剂可以缓解一些问题，但它们可能会损害识别系统 自己此外，变性剂仅去除蛋白质的一些分子内相互作用。 Glyphic Biotechnologies正在开发一种新的策略， 从一个异质的样品中。该过程基于将N-末端氨基酸连接到可裂解的化学键上。 化学连接剂，随后将其局部地束缚到表面。接头的切割除去N-末端 蛋白质中的氨基酸进行高灵敏度鉴定，不受蛋白质结构或氨基酸的干扰。 %的氨基酸。对每个后续氨基酸重复该过程，产生蛋白质序列。的 这种方法可以在数小时内同时对数百万到数十亿个蛋白质分子进行测序， 通过使大规模蛋白质测序可行、廉价和常规，彻底改变蛋白质分析。 目前的建议集中在开发特异性检测蛋白质N-末端氨基酸的试剂， 允许通过这种N-末端分离策略数字化鉴定氨基酸。在目标1中，我们将生成 抗体识别至少10种不同的分离氨基酸-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，并证明使用 他们在未修饰的蛋白质背景中对单个蛋白质进行测序。 这些目标的成功将使Glyphic蛋白测序平台能够检测、定量和测序 以无偏的方式在复杂蛋白质混合物中的单个蛋白质-而无需任何关于其身份的先验知识 甚至他们的存在。当商业化时，它将使基于蛋白质的疾病临床诊断成为可能 存在于患者样品中，并允许鉴定独特的蛋白质以用于迄今未知的生物标志物。