Ladner Yoann, Coussot Gaelle, Ebner Stefanie, Ibrahim Amal, Vidal Laetitia, Perrin Catherine
Institut des Biomolécules Max Mousseron-IBMM, Centre National de la Recherche Scientifique, Université de Montpellier, Unité Mixte de Recherche 5247, Faculté de Pharmacie, Montpellier cedex 5, France.
GemacBio, Lieu-dit Berganton, Saint-Jean d'Illac, France.
Electrophoresis. 2016 Jan;37(2):256-66. doi: 10.1002/elps.201500349. Epub 2015 Dec 9.
This work aims at studying the optimization of an on-line capillary electrophoresis (CE)-based tryptic digestion methodology for the analysis of therapeutic polypeptides (PP). With this methodology, a mixture of surrogate peptide fragments and amino acid were produced on-line by trypsin cleavage (enzymatic digestion) and subsequently analyzed using the same capillary. The resulting automation of all steps such as injection, mixing, incubation, separation and detection minimizes the possible errors and saves experimental time. In this paper, we first study the differents parameters influencing PP cleavage inside the capillary (plug length, reactant concentration, incubation time, diffusion and electrophoretic plugs mixing). In a second part, the optimization of the electrophoretic separation conditions of generated hydrolysis products (nature, pH and ionic strength (I) of the background electrolyte (BGE)) is described. Using the optimized conditions, excellent repeatability was obtained in terms of separation (migration times) and proteolysis (number of products from enzymatic hydrolysis and corresponding amounts) demonstrating the robustness of the proposed methodology.
这项工作旨在研究一种基于在线毛细管电泳（CE）的胰蛋白酶消化方法用于治疗性多肽（PP）分析的优化。采用这种方法，通过胰蛋白酶裂解（酶解）在线生成替代肽片段和氨基酸的混合物，随后使用同一根毛细管进行分析。进样、混合、孵育、分离和检测等所有步骤实现自动化，最大限度地减少了可能出现的误差并节省了实验时间。在本文中，我们首先研究影响毛细管内PP裂解的不同参数（塞长度、反应物浓度、孵育时间、扩散和电泳塞混合）。在第二部分，描述了对生成的水解产物进行电泳分离条件的优化（背景电解质（BGE）的性质、pH和离子强度（I））。使用优化后的条件，在分离（迁移时间）和蛋白水解（酶解产物的数量及相应含量）方面获得了出色的重复性，证明了所提出方法的稳健性。
