應用液相層析串聯質譜儀定量分析研究細胞穿透胜–ECP10

Abstract

細胞穿透胜肽 (cell-penetrating peptides，CPPs) 是描述由三十個氨基酸以下所組成，並具有細胞穿透活性的多肽。物質分子藉由與細胞穿透胜肽的結合，可克服原生質膜的阻礙而進入細胞內部，是現今藥物傳送醫療最有發展潛力的一項工具。嗜酸性白血球陽離子蛋白 (eosinophil cationic protein，ECP) 序列中的「32NYRWRCKNQN41」，具有進入細胞的活性能力，為一新的細胞穿透胜肽。將此序列合成並修飾上螢光物質後 (F-ECP10)，即可進行各式實驗以觀測其穿透細胞之狀況與效果。我們利用液相層析串聯質譜儀的技術，針對F-ECP10的作用及偵測進行一系列探討。首先，我們發現所使用的RPMI 1640細胞培養基成份中，胱胺酸參與了此作用機制，由質譜鑑定結果，F-ECP10與胱胺酸產生雙硫鍵交換的反應，以致實際基質中，F-ECP10是與半胱胺酸以雙硫鍵結合的形式存在 (F-ECP10-Cys)，推測實際上與細胞作用之分子應為F-ECP10-Cys構形之分子。因此，我們針對其進行一系列層析及質譜儀的最佳化參數設定，並以細胞培養液製作定量校正曲線的探討。可得到其線性方程式R2值0.9985，濃度的線性範圍為50 nM至10 μM，偵測極限為6.29 nM，且具有良好的再現性。在此我們利用液相層析串聯質譜儀技術，發展出一套針對F-ECP10定量偵測的研究方式，操作簡便且省時，未來可應用於細胞穿透胜肽或其他多肽之偵測與應用。

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.