Single-molecule protein sequencing by barcoding of N-terminal amino acids

通过 N 端氨基酸条形码进行单分子蛋白质测序

• 批准号: 10757309
• 负责人: Daniel Masao Estandian
• 金额: $ 35.9万
• 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: 2023-09-15 至 2024-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10757309
• 关键词: Acceleration; Acids; Address; Affect; Affinity; Amino Acid Sequence; Amino Acids; Antibodies; Bar Codes; Basic Science; Binding; Biological; Biological Markers; Biology; Biotechnology; Blood; Buffers; C-terminal; Cells; Charge; Chemicals; Chemistry; Complex; Complex Mixtures; Coupled; DNA; DNA sequencing; Data; Detection; Disease; Face; Functional disorder; Future; Gel; Genome; Growth and Development function; High Pressure Liquid Chromatography; High-Throughput Nucleotide Sequencing; Hour; Individual; Libraries; Light; Link; Mass Spectrum Analysis; Methods; N-terminal; Parents; Peptide Mapping; Peptide Sequence Determination; Peptides; Polymerase; Positioning Attribute; Process; Protein Analysis; Protein Secretion; Protein Sequence Analysis; Proteins; Proteome; Proteomics; RNA; Reaction; Reagent; Research; Resolution; Rest; Sampling; Side; Signal Transduction; Specificity; Structure; System; Technology; base; clinical diagnostics; commercialization; cost effective; drug development; gel electrophoresis; improved; innovation; magnetic beads; molecular mass; nanopore; next generation; next generation sequencing; novel; pathogen; protein function; single molecule; transcriptome

SUMMARY Proteins are responsible for much of the structure and function of all cells. Subtle changes in expression of var- ious protein forms are critical for proper growth and development, but irregularities can cause deleterious cellular effects or large-scale biological dysfunction. Sequencing samples with both high- and low-abundance proteins could greatly accelerate research into protein function and biology, but there is currently no efficient and cost-effective strategy to sequence mixtures of unknown protein molecules at single-amino-acid resolution. Two methods are commercially available for protein sequencing. The first method, “Edman degradation”, re- quires purification of the individual target protein. Bulk quantities of whole protein or purified fragments are sequenced by cleaving off the first (N-terminal) amino acid and chemically identifying it. The second method, based on mass spectrometry, requires enzymatically degrading a single protein or mixture of proteins into small fragments, then analyzing the molecular mass and charge of each fragment. This information is com- pared to that of known protein sequences to infer the identity of the input proteins. Both of these methods require ~1 million molecules of each protein for detection. Currently, Edman degradation cannot be used on heterogeneous protein mixtures, further limiting its utility. Single molecule protein sequencing is hindered by the number and diversity of amino acids, as well as the in- teractions between amino acids that interfere with chemical identification of their side chains. Identifying N- terminal amino acid that is still attached to the rest of the protein will be hindered by the N-1 (and N-2) amino acids, proportional to the bulk of the side chain. Harsh denaturation agents can mitigate some of these issues. However, these reagents can compromise the biomolecule-based identification systems themselves and do not fully remove the steric hindrance, affecting the access to the N-terminal amino acid. Glyphic Biotechnologies has developed a novel “Next-Generation” protein sequencing strategy, in which DNA barcodes associate rounds of cleaved N-terminal amino acid with a protein-specific barcode. Each of the 20 different amino acids will be first cleaved (circumventing the stearic hindrance of the N-1 amino acid) and then captured by specific antibodies. Each amino acid will then be associated with two barcodes, indicating the (1) originating protein and (2) sequential position this amino acid can be found in. After next-generation DNA se- quencing of all conjugated barcodes, this information can be deconvoluted – placing each amino acid into the correct position within the correct protein. This approach has the potential to be scaled to sequence millions to billions of single molecules simultaneously in hours. Developing this technology will revolutionize protein analysis by making large-scale protein sequenc- ing feasible, inexpensive, and routine.

概括 蛋白质负责所有细胞的大部分结构和功能。 var-表达的细微变化 IOUS蛋白质形式对于适当的生长和发展至关重要，但不规则性会导致有害 细胞效应或大规模生物功能障碍。具有高充值和低含量的样品测序样品 蛋白质可以大大加速蛋白质功能和生物学的研究，但目前尚无高效 以及在单氨基酸分辨率下未知蛋白质分子的混合物的成本有效策略。 两种方法可用于蛋白质测序。第一种方法，“埃德曼退化”，重新 需要纯化单个靶蛋白。大量的全蛋白或纯化片段是 通过切割第一（N末端）氨基酸并化学识别它来测序。第二种方法， 基于质谱法，需要酶促降解单个蛋白质或蛋白质的混合物 小片段，然后分析每个片段的分子质量和电荷。此信息是 涉及已知蛋白序列的序列，以推断输入蛋白的身份。这两种方法 每种蛋白质需要约100万个分子进行检测。目前，Edman退化不能用于 异质蛋白混合物，进一步限制了其效用。 单分子蛋白测序受到氨基酸的数量和多样性的阻碍 氨基酸之间干扰其侧链的化学鉴定的氨基酸。识别n- 仍附着在其余蛋白质上的末端氨基酸将受到N-1（和N-2）氨基的阻碍 酸，与大部分侧链成比例。苛刻的变性代理可以减轻其中一些问题。 但是，这些试剂可以损害基于生物分子的识别系统本身并做 不能完全去除空间障碍，从而影响进入N末端氨基酸的通道。 甘氨酸生物技术已经开发了一种新型的“下一代”蛋白测序策略，其中DNA 条形码将裂解的N末端氨基酸与蛋白质特异性条形码相关。 20个 首先将裂解不同的氨基酸（绕过N-1氨基酸的蒸汽障碍），然后 由特定抗体捕获。然后，每个氨基酸将与两个条形码相关联，表明（1） 起源蛋白质和（2）顺序位置可以在下一代DNA之后找到该氨基酸。 淬灭所有共轭条形码，可以将这些信息解卷 - 将每个氨基酸放入 正确的位置在正确的蛋白质中。 这种方法有可能简单地缩放到数百万到数十亿个单分子的序列 数小时。开发该技术将通过制作大规模蛋白质序列来彻底改变蛋白质分析。 可行，廉价和常规。