Wainaina Moses N, Shibata Takayuki, Smanmoo Chaivat, Kabashima Tsutomu, Kai Masaaki
Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Bunkyo-Machi 1-14, Nagasaki 852-8521, Japan.
Anal Biochem. 2008 Mar 15;374(2):423-5. doi: 10.1016/j.ab.2007.12.003. Epub 2007 Dec 7.
A modified Edman degradation method where fluorescent derivatives of amino acids were generated from the postcleavage products of a peptide is described. In the method, the target peptide was applied onto double glass fiber membranes in a small filter disk (4 mm i.d.) and then treated with small amounts of reagents for the manual sequencing of the peptide. The anilinothiazolinone (ATZ) of N-terminus amino acid residue after the isolation from the solid-phase membranes was reacted with a primary amine, 4-(1'-cyanoisoindolyl)aniline (CIA), to form a more stable and sensitive fluorescent derivative, phenylthiocarbamoyl-CIA. An average yield of 85% was obtained in neutral pH conditions for the CIA reaction. The ATZ-CIA-amino acids were separated by reversed-phase liquid chromatography and detected by fluorometry. The lower limits of the detection for amino acids after the Edman degradation were 0.16 to 0.52 pmol (signal/noise ratio=3) on the column. The sensitivity was approximately 10 times higher than ultraviolet absorbance detection of phenylthiohydantoin products in the conventional Edman degradation. The suitability of the method was demonstrated by the sensitive manual sequencing of insulin chain B composed of 30 amino acids.
本文描述了一种改良的埃德曼降解法，该方法从肽的切割后产物中生成氨基酸的荧光衍生物。在该方法中，将目标肽应用于小滤盘（内径4 mm）中的双层玻璃纤维膜上，然后用少量试剂进行肽的手动测序。从固相膜上分离出的N端氨基酸残基的苯氨基噻唑啉酮（ATZ）与伯胺4-(1'-氰基异吲哚基)苯胺（CIA）反应，形成更稳定、更灵敏的荧光衍生物苯硫代甲酰基-CIA。在中性pH条件下，CIA反应的平均产率为85%。ATZ-CIA-氨基酸通过反相液相色谱分离，并用荧光法检测。埃德曼降解后氨基酸的检测下限在柱上为0.16至0.52 pmol（信噪比=3）。该灵敏度比传统埃德曼降解中苯硫代乙内酰脲产物的紫外吸收检测高约10倍。由30个氨基酸组成的胰岛素B链的灵敏手动测序证明了该方法的适用性。
