DEVELOPMENT OF METHODS FORC-TERMINAL SEQUENCING OF PEPTIDES AND PROTEINS

一种简便、有效的测定氨基末端的方法 长期以来，人们一直在寻找蛋白质的氨基酸序列。一件有希望的事 方法利用多肽制剂的质谱分析 通过用酶降解感兴趣的多肽而产生 羧基肽酶，其中测量的质量的差异 降解产物定义了羧基末端序列。然而， 这种方法的实用性受到了巨大差异的限制。 在不同末端氨基酸残基的消化速度中， 导致产生不间断序列定义的困难 多肽纤维。为了确保不间断地混合 定义序列的多肽成分(即，连续的多肽 阶梯)，有必要要么缩小差别税率 去除末端氨基酸或呈现每个肽的一小部分 产品耐细微降解性。对于这一目标，我们 探索了一种新的策略，在这种策略中，水解和氨解是 被设置为由相同的外肽酶催化的竞争反应。这个 水解反应去除末端氨基酸残基，而 氨解(反向蛋白质分解)反应旨在增加一个 终点组到新设计的终点站。我们用一种氨基 酸酰胺(赖氨酰胺)作为终止剂，因为它竞争 有效地作为酰基酶的亲核剂和水 中间体，因为生成的肽酰胺相对 耐水解性的。我们的结果表明，动力学效应 由于添加了大量摩尔过量的赖氨酰胺，可以 大大改善了对羧基肽酶消化的控制 羧基末端测序的质谱学读数 得到的多肽梯形图。酶消化的差异很大 速率趋于均匀，因为水解率和氨解率都是一样的 都是由酶催化的。尽管FIND-DIER优化是 可取的是，目前的战略有可能提供一个容易的 一种获得有限羧基末端序列的可靠方法 多肽和蛋白质。以协助进行c-末端测序 通过上面概述的方法，我们也开始开发出一种 从Lys-C中分离C-端肽的实用方法 蛋白质的消化(见下列子项目)。

Facile, effective means for determining the caThoxyl-terminal amino acid sequence of proteins have long been sought. One promising approach utilizes mass spectrometric analysis of peptide fi-agments produced by enzymatic degradation of the polypeptide of interest with carboxypeptidases, wherein differences in the measured masses of the degradation products define the carboxyl-terminal sequence. However, the utility of this approach has been limited by large discrepancies in the rates of digestion of different terminal amino acid residues, leading to difficulties in producing uninterrupted sequence-defining peptide firagments. In order to ensure a mixture of uninterrupted sequence-defining peptide ftagrnents (i.e., a continuous "peptide ladder), it is necessary to either diminish differential rates of terminal amino acid removal or to render a fraction of each peptide product resistant to finther degradation. Towards this ob ective, we have explored a new strategy in which hydrolysis and aminolysis are set up as competing reactions catalyzed by the same exopeptidase. The hydrolysis reaction removes the terminal amino acid residue, while the aminolysis (reverse proteolysis) reaction is designed to add a terminating group to the newly fonned termini. We use an amino acid-amide (lysinamide) as a terminating reagent because it competes effectively as a nucleophile with water for the acyl-enzyme intermediate and because the resulting peptide amide is relatively resistant to hydrolysis. Our results demonstrate that kinetic effects resulting from the addition of a large molar excess of lysinamide can considerably improve the control of carboxypeptidase digestion for carboxyl-terminal sequencing by mass spectrometric readout of the resulting peptide ladders. Large discrepancies in enzyme digestion rates tend to be evened out because both hydrolysis and arninolysis are catalyzed by the enzyme. Although fin-dier optimization is desirable, the present strategy has the potential to provide an easy and reliable method for obtaining limited carboxyl-terminal sequences of peptides and proteins. To assist in the c-terminal sequencing of proteins by the method outlined above, we have also begun to develop a practical means for isolating the C-terminal peptide from a lys-C digest of a protein (see following subproject).