Quantitative Analysis of Cell-penetrating Peptide–ECP10 by Liquid Chromatography/Mass Spectrometry

• Main Author: 鄒函文
• Other Authors: 謝有容
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/2b95et

碩士 === 國立交通大學 === 應用化學系碩博士班 === 101 === Cell-penetrating peptide (CPP), usually less than 30 amino acids, was used to describe peptides that have ability to penetrate cell membranes and translocate different cargos into cells. CPPs were considered to hold great potential for delivery of therapeutic molecules. Eosinophil cationic protein (ECP) was a well-known biomarker for asthma and other airway inflammation. A 10 amino acid sequence 32NYRWRCKNQN41 (ECP10) in ECP was found that existed cell penetrating property and studied in this article. To investigate the mechanism of internalization and quantitative analysis of F-ECP10, we used the technique of liquid chromatography tandem mass spectrometry (LC/MS) for our studies. We found the cystine contained in the medium RPMI 1640 was reacted with F-ECP10 forming derivative. By the confirmation of mass spectrometry, F-ECP10 and cystine underwent disulfide exchange, and formed the disulfide bond between ECP10 and cysteine (F-ECP10-Cys). We supposed it is the structure of F-ECP10-Cys that interacted with cells. Therefore, we optimized parameters based on LC/MS and plotted the calibration curve in RPMI 1640 medium for F-ECP10-Cys. The linear range was from 50 nM to 10 μM with the detection limit 6.29 nM. By utilizing the technique of LC/MS here, we developed a new method for F-ECP10 to study the mechanism of internalization and quantitative detection. It was simple, time-saving, and good for further applications on the fields of cell-penetrating peptide.