SEQUENCING N-TERMINALLY BLOCKED PROTEINS

蛋白质和多肽的自动埃德曼降解至关重要 提供氨基酸序列信息用于识别和识别的重要性 对包含相应结构基因的DNA克隆进行测序。这个 包括蛋白质/多肽样品制备的复杂程序， 测序化学本身和PTH氨基酸鉴定已经成为 不断改进以提高灵敏度的高科技 和适用范围，所以低丰度和低丰度的蛋白质 可以分析日益多样化的属性。目前， 这项技术最重要的限制是大约80%的 蛋白质通过Nα-酰化被阻止埃德曼降解 N-末端残基。这一限制的重要性源于 从N末端获得的序列信息的显著重要性 用于基因克隆和测序研究的完整蛋白质。虽然 内部氨基酸序列信息通常可以从 被阻断的蛋白质，它的获取总是消耗相当大的 与N-末端序列分析相比，蛋白质的量更多，并且缺乏 N-末端序列随后使DNA序列数据更加困难 获得并解释。 本提案旨在建立解除皮考莫阻塞的一般方法。 蛋白质的量，以便高灵敏度的氨基酸测序 用于确定N-末端序列。渲染小尺寸的过程 易被特定解锁酶附着的蛋白质的量 将寻求在工作蛋白质化学中实施 实验室。该程序将基于共价固定化 与埃德曼兼容的固体载体上的底物蛋白质 测序。尽管潜在地适合Na-乙酰化氨基酸 已知的水解物会破坏小肽底物， 提议的风险很高，因为没有证据表明它们会持续更长时间 多肽。然而，该项目的成功将导致很大的 氨基酸序列分析灵敏度的提高 大多数蛋白质，并将代表着一个重大的进步 这项技术在分子生物学中的应用。

Automated Edman degradation of proteins and peptides is of crucial importance in providing amino acid sequence information for identifying and sequencing DNA clones that contain the corresponding structural genes. The complex of procedures encompassing protein/peptide sample preparation, the sequencing chemistry itself, and PTH amino acid identification, has become a high technology that is constantly being modified to improve sensitivity and range of applicability, so that proteins of lower abundance and increasingly diverse properties can be analyzed. At the present time, the most important limitation to the technology is that approximately 80% of proteins are blocked tot he Edman degradation by Nalpha- acylation of the N-terminal residue. The significance of this limitation derives from the preeminent importance of sequence information acquired from the N-terminus of the intact protein for gene cloning and sequencing studies. Although internal amino acid sequence information can generally be acquired from blocked proteins, its acquisition always consumes considerably larger amounts of protein than an N-terminal sequence analysis, and the lack of an N-terminal sequence subsequently makes the DNA sequence data more difficult to obtain and interpret. This proposal seeks to establish general methods for deblocking picomole amounts of proteins so that high sensitivity amino acid sequencing can be used to determine the N-terminal sequence. A procedure for rendering small amounts of protein susceptible to attach by specific deblocking enzymes will be sought for implementation in a working protein chemistry laboratory. The procedure will be based on covalent immobilization of the substrate protein on a solid support that is compatible with Edman sequencing. Although potentially suitable Nalpha - acetylation amino acid hydrolyses are known that will deblock small peptide substrates, the proposal is high risk because they have not been shown to work on longer polypeptides. However, success of the project would result in a large improvement in the sensitivity of amino acid sequence analysis of the majority of proteins and would represent a major improvement in the usefulness of the technology in molecular biology.