Fischer P M, Howden M E
Division of Biological and Health Sciences, Deakin University, Geelong, Victoria, Australia.
Anal Biochem. 1989 Feb 15;177(1):46-9. doi: 10.1016/0003-2697(89)90011-0.
A single-column high-performance liquid chromatographic separation of 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin amino acid derivatives, generated during polypeptide sequence analysis by the 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate/phenylisothiocyanate double coupling technique, is described. Recovery of the serine and threonine derivatives was improved by substituting boron trifluoride-diethyl etherate for trifluoroacetic acid in the thiazolinone cleavage reactions. Residues, including the S-carboxymethyl derivative of cysteine, were assigned after a single injection and a cycle time of 30 min. Quantities of 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin amino acid derivatives as low as 100 fmol were detected. Interference of sequencing artefacts with residue assignment was avoided. This technique allows simultaneous manual sequencing of several proteins or peptides at the level of a few picomoles.
本文描述了一种单柱高效液相色谱法，用于分离在多肽序列分析过程中，通过4-N,N-二甲基氨基苯偶氮苯4'-异硫氰酸酯/苯异硫氰酸酯双重偶联技术生成的4-N,N-二甲基氨基苯偶氮苯4'-硫代乙内酰脲氨基酸衍生物。在噻唑啉酮裂解反应中，用三氟化硼-乙醚络合物替代三氟乙酸，提高了丝氨酸和苏氨酸衍生物的回收率。单次进样和30分钟的循环时间后，可对包括半胱氨酸的S-羧甲基衍生物在内的残基进行鉴定。检测到低至100飞摩尔的4-N,N-二甲基氨基苯偶氮苯4'-硫代乙内酰脲氨基酸衍生物。避免了测序假象对残基鉴定的干扰。该技术允许在几皮摩尔水平上同时手动测序几种蛋白质或肽。
