Bailey J M, Shenoy N R, Ronk M, Shively J E
Beckman Research Institute of the City of Hope, Division of Immunology, Duarte, California 91010.
Protein Sci. 1992 Jan;1(1):68-80. doi: 10.1002/pro.5560010108.
Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS-ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145-3156). The current study is a continuation of this work and describes the construction of an instrument for automated C-terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58-67), the use of sodium trimethylsilanolate as a novel reagent for the specific cleavage of the derivatized C-terminal amino acid, and the development of methodology to sequence through the difficult amino acid, aspartate. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS-ITC, and cleavage of the derivatized C-terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on-line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids were found to sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not be capable of derivatization. In spite of these current limitations, the methodology should be a valuable new tool for the C-terminal sequence analysis of peptides.
蛋白质和肽可以使用异硫氰酸酯试剂从羧基末端进行测序，以生成氨基酸硫代乙内酰脲衍生物。我们实验室之前的研究集中在溶液相条件下，用于用三甲基硅烷异硫氰酸酯（TMS-ITC）形成肽基硫代乙内酰脲，以及将这些肽基硫代乙内酰脲水解为氨基酸硫代乙内酰脲衍生物和能够继续降解的新的缩短肽（Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145 - 3156）。当前的研究是这项工作的延续，描述了用于自动C末端测序的仪器的构建、硫氰酸酯化学在与新型聚乙烯固体支持物共价偶联的肽上的应用（Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58 - 67）、使用三甲基硅醇钠作为用于特异性切割衍生化C末端氨基酸的新型试剂，以及开发用于对困难氨基酸天冬氨酸进行测序的方法。描述了用于与羧酸修饰的聚乙烯共价连接的肽的C末端测序的自动化程序。该化学过程包括用乙酸酐活化、用TMS-ITC衍生化，以及用三甲基硅醇钠切割衍生化的C末端氨基酸。通过使用Phenomenex Ultracarb 5 ODS(30)柱和含有戊烷磺酸的三乙胺/磷酸缓冲系统的在线高效液相色谱法鉴定硫代乙内酰脲氨基酸。通过对包含所有20种常见氨基酸的模型肽进行测序，检验了我们的自动C末端测序方法的通用性。发现除了天冬酰胺和天冬氨酸只能部分去除，以及脯氨酸不能进行衍生化外，所有氨基酸的测序产率都很高（90%或更高）。尽管存在这些当前的局限性，该方法对于肽的C末端序列分析应该是一种有价值的新工具。
