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

总结 蛋白质负责所有细胞的大部分结构和功能。变异表达的细微变化- 各种蛋白质形式对正常生长和发育至关重要，但不规则性可导致有害的 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 蛋白质可以大大加速对蛋白质功能和生物学的研究，但目前还没有有效的方法。 和具有成本效益的策略，以单氨基酸分辨率对未知蛋白质分子的混合物进行测序。 有两种方法可用于蛋白质测序。第一种方法，“埃德曼降解”，重新- 需要纯化单个靶蛋白。批量的全蛋白或纯化的片段是 通过切下第一个（N-末端）氨基酸并对其进行化学鉴定来测序。第二种方法， 基于质谱法，需要将单一蛋白质或蛋白质混合物酶促降解成 小片段，然后分析每个片段的分子量和电荷。这些信息是COM- 与已知蛋白质序列的相似性来推断输入蛋白质的身份。这两种方法 每种蛋白质需要约100万个分子用于检测。目前，Edman降解不能用于 异质蛋白质混合物，进一步限制了其效用。 单分子蛋白质测序受到氨基酸的数量和多样性的阻碍，以及蛋白质的内部结构。 干扰其侧链化学鉴定的氨基酸之间的相互作用。识别N- 仍然连接到蛋白质其余部分的末端氨基酸将被N-1（和N-2）氨基阻碍 酸，与侧链的体积成比例。苛刻的变性剂可以缓解其中一些问题。 然而，这些试剂可能损害基于生物分子的识别系统本身， 不能完全除去空间位阻，影响N-末端氨基酸的进入。 Glyphic Biotechnologies开发了一种新的“下一代”蛋白质测序策略，其中DNA 条形码将切割的N-末端氨基酸的轮与蛋白质特异性条形码相关联。的20 不同的氨基酸将首先被切割（避开N-1氨基酸的位阻），然后 被特定的抗体捕获然后每个氨基酸将与两个条形码相关联，指示（1） 起始蛋白质和（2）可以发现该氨基酸的顺序位置。在下一代DNA测序之后， 通过对所有缀合的条形码进行测序，可以将该信息解卷积-将每个氨基酸置于缀合的条形码中。 正确蛋白质内的正确位置。 这种方法有可能被扩展到同时测序数百万到数十亿个单分子 几小时后开发这项技术将彻底改变蛋白质分析，使大规模的蛋白质测序， 可行的，便宜的，常规的。

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.