Matsueda G R, Haber E, Margolies M N
Biochemistry. 1981 Apr 28;20(9):2571-80. doi: 10.1021/bi00512a032.
Quantitative solid-phase Edman degradation was used for the amino acid sequence analysis of synthetic peptidyl-resins prepared by the Merrifield solid-phase procedure. A model peptide, Ala-[3H]Pro-Ala-Gly-Phe-Ala-Gly-, was synthesized on a solid support and was sequenced to measure the efficiency of the solid-phase sequencing protocol used. An average of 92% of the first four residues was removed from the peptidyl-resin as indicated by subtractive amino acid analysis. Quantitation of the radioactive proline residue at cycle 2 revealed that it was efficiently recovered both from the acid conversion procedure (99%) and also following high-pressure liquid chromatography of the phenylthiohydantoin (Pth) amino acid (88%). In order to facilitate identification and quantification of the side chain protected Pth amino acids, we prepared these derivatives and characterized them by high-pressure liquid chromatography. Thereafter, by the use of solid-phase Edman degradation as an analytical procedure, the synthesis of residues 2-118 of the heavy-chain variable region (VH) of a homogeneous rabbit antibody was undertaken. At 10-15-residue intervals during the solid-phase synthesis, samples of peptidyl-resin were removed from the synthesis vessel and sequenced. When gross synthetic errors caused by deletion of amino acids residues were detected, the solid-phase synthesis was terminated and restarted by using modified protocols. A 117-residue peptidyl-resin was prepared finally which possessed the desired amino acid sequence as indicated by a series of solid-phase Edman degradation experiments. In the final degradation experiment on the 117-residue peptidyl-resin, a 92% efficiency for the automatic Edman reaction was measured ([3H]Leu, penultimate amino-terminal residue). We have found two advantages for the concurrent use of solid-phase Edman degradation during an extended solid-phase synthesis: (1) on the basis of the level of error due to incomplete incorporation of amino acids, the solid-phase assembly could be terminated in favor of restarting the synthesis, hence avoiding further work on a defective product and (2) direct verification of incorporation of amino acids, which during acid hydrolysis are destroyed (Cys, Trp) or are deamidated (Asn, Gln), is possible by high-pressure liquid chromatography of the corresponding Pth derivatives.
定量固相埃德曼降解法用于对通过梅里菲尔德固相法制备的合成肽基树脂进行氨基酸序列分析。在固相载体上合成了一个模型肽Ala-[³H]Pro-Ala-Gly-Phe-Ala-Gly-，并对其进行测序以测定所使用的固相测序方案的效率。通过减去氨基酸分析表明，平均92%的前四个残基从肽基树脂上被去除。对第2轮循环中的放射性脯氨酸残基进行定量分析表明，它在酸转化过程中（99%）以及随后的苯硫基乙内酰脲（Pth）氨基酸的高压液相色谱分析后（88%）均能有效回收。为了便于对侧链受保护的Pth氨基酸进行鉴定和定量，我们制备了这些衍生物并通过高压液相色谱对其进行表征。此后，通过使用固相埃德曼降解作为一种分析方法，进行了一种同源兔抗体重链可变区（VH）第2至118位残基的合成。在固相合成过程中，每隔10 - 15个残基，从合成容器中取出肽基树脂样品并进行测序。当检测到因氨基酸残基缺失导致的总体合成错误时，固相合成终止，并使用改进方案重新开始。最终制备了一种117个残基的肽基树脂，一系列固相埃德曼降解实验表明其具有所需的氨基酸序列。在对117个残基的肽基树脂进行的最终降解实验中，自动埃德曼反应的效率为92%（[³H]Leu，倒数第二个氨基末端残基）。我们发现了在长时间固相合成过程中同时使用固相埃德曼降解的两个优点：（1）基于由于氨基酸不完全掺入导致的错误水平，固相组装可以终止以便重新开始合成，从而避免对有缺陷的产物进行进一步操作；（2）通过相应Pth衍生物的高压液相色谱分析，可以直接验证氨基酸的掺入情况，这些氨基酸在酸水解过程中会被破坏（Cys、Trp）或脱酰胺（Asn、Gln）。
