Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Bunkyo-Machi 1-14, Nagasaki 852-8521, Japan.
J Chromatogr A. 2011 Jun 17;1218(24):3757-62. doi: 10.1016/j.chroma.2011.04.040. Epub 2011 Apr 21.
A method for sequence analysis and identification of phosphoamino acids in peptides based on high performance liquid chromatography (HPLC) is described. The peptides were derivatized with an Edman type reagent, 4-(1'-cyanoisoindolyl)phenylisothiocyanate (CIPIC) and subsequently cleaved to generate stable and fluorescent 4-(1'-cyanoisoindolyl)phenylthiazolinone (CIP-TZ)-amino acids. Several experimental factors that affected derivatization on membranes were examined. Under the optimized conditions, the CIP-TZ derivatives of Try(p), Thr(p) and Ser(p) were obtained and separated from their parent amino acids with baseline resolution using an isocratic elution system. Up to the 4th residue of phosphorylated pentapeptides was successfully identified, whereas phosphoamino acid residues could not be detected by the conventional procedure using phenylisothiocyanate (PITC). The results demonstrated the potential of CIPIC as a derivatization reagent for peptide sequencing and the applicability of the method for the study and identification of phosphoamino acids in peptides.
本文描述了一种基于高效液相色谱（HPLC）的用于分析和鉴定肽中磷酸氨基酸的方法。该方法通过 Edman 型试剂 4-(1'-氰基异吲哚基)苯基异硫氰酸酯（CIPIC）对肽进行衍生化，然后进行裂解，生成稳定且具有荧光性的 4-(1'-氰基异吲哚基)苯基噻唑啉酮（CIP-TZ）-氨基酸。本研究考察了影响膜上衍生化的几个实验因素。在优化条件下，成功获得了 Try(p)、Thr(p)和 Ser(p)的 CIP-TZ 衍生物，并采用等度洗脱系统将其与母体氨基酸基线分离。该方法可成功鉴定至四肽的磷酸化残基，而传统的使用苯异硫氰酸酯（PITC）的方法则无法检测到磷酸氨基酸残基。结果表明，CIPIC 可作为一种用于肽测序的衍生化试剂，该方法适用于研究和鉴定肽中的磷酸氨基酸。
