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表达的微妙变化。 丰富的蛋白质形式对于正常的生长和发育至关重要，但不规则的蛋白质形式可能会导致有害的后果 细胞效应或大规模生物功能障碍对高丰度和低丰度样品进行测序。 蛋白质可以极大地加速蛋白质功能和生物学的研究，但目前还没有有效的方法 以及以单氨基酸分辨率对未知蛋白质分子混合物进行测序的经济有效的策略。 商业上有两种方法可用于蛋白质测序，第一种方法是“Edman 降解”。 需要纯化大量的完整蛋白质或纯化片段。 通过切除第一个（N 端）氨基酸并进行化学鉴定来进行测序第二种方法， 基于质谱分析，需要酶促地将单一蛋白质或蛋白质混合物降解为 小碎片，然后分析每个碎片的分子质量和电荷。 与已知蛋白质序列进行比较以推断输入蛋白质的身份。 每种蛋白质需要约 100 万个分子才能进行检测。目前，Edman 降解法无法用于检测。 异质蛋白质混合物，进一步限制了其用途。 单分子蛋白质测序受到氨基酸数量和多样性以及内部结构的阻碍。 氨基酸之间的相互作用会干扰其侧链的化学鉴定。 仍与蛋白质其余部分相连的末端氨基酸将受到 N-1（和 N-2）氨基酸的阻碍 与侧链的体积成比例的强去饱和剂可以缓解其中一些问题。 然而，这些试剂可能会损害基于生物分子的识别系统本身，并且 未完全消除空间位阻，影响N端氨基酸的进入。 Glyphic Biotechnologies 开发了一种新颖的“下一代”蛋白质测序策略，其中 DNA 条形码将切割的 N 端氨基酸与 20 个蛋白质特异性条形码中的每一个相关联。 不同的氨基酸将首先被切割（规避 N-1 氨基酸的空间位阻），然后 然后，每个氨基酸将与两个条形码相关联，表示 (1) 原始蛋白质和 (2) 该氨基酸的连续位置。下一代 DNA 分离后 对所有共轭条形码进行测序后，可以对该信息进行解卷积——将每个氨基酸放入 正确蛋白质内的正确位置。 这种方法有可能扩大规模，同时对数百万至数十亿个单分子进行测序 开发这项技术将通过大规模蛋白质序列彻底改变蛋白质分析。 可行、廉价且常规。