Institut des Biomolécules Max Mousseron (IBMM, UMR 5247 CNRS, Université de Montpellier, Ecole Nationale Supérieure de Chimie de Montpellier), Faculté de Pharmacie, 34093 Montpellier Cedex 5, France.
Institut des Biomolécules Max Mousseron (IBMM, UMR 5247 CNRS, Université de Montpellier, Ecole Nationale Supérieure de Chimie de Montpellier), Faculté de Pharmacie, 34093 Montpellier Cedex 5, France.
Talanta. 2019 Feb 1;193:146-151. doi: 10.1016/j.talanta.2018.09.090. Epub 2018 Sep 26.
The main purpose of the present work is to provide a fully integrated temperature control of in-line tryptic digestion in order to facilitate the quality control of polypeptidic therapeutic compounds. The in-line enzymatic reaction was performed in 100 mM bicarbonate ammonium whereas a mixture of citric acid/ε-amino caproic acid (pH 5.0 and I 75 mM) was used as a background electrolyte (BGE). After the injection of all reactants (substrate, enzyme, proteolysis buffer), a BGE plug was injected to push all reactants until a position where the capillary is thermostated. Then, the enzymatic reaction was initiated during 15 min of incubation time and finally, a voltage was applied to separate the generated proteolysis products. The methodology was developped regarding the effects of the BGE plug length and pressure on the reactants plug mixing and on the advanced of the tryptic digestion. Successful temperature control of in-line proteolysis with excellent repeatability was obtained in optimal cleavage and separation conditions.
本工作的主要目的是提供在线胰蛋白酶消化的完全集成温度控制，以方便治疗性多肽化合物的质量控制。在线酶反应在 100mM 碳酸氢铵铵中进行，而柠檬酸/ε-氨基己酸（pH5.0 和 I75mM）混合物用作背景电解质（BGE）。在注入所有反应物（底物、酶、蛋白水解缓冲液）后，注入 BGE 塞以推动所有反应物，直到毛细管恒温的位置。然后，在 15 分钟孵育时间内启动酶反应，最后施加电压以分离生成的蛋白水解产物。该方法是针对 BGE 塞长度和压力对反应物塞混合以及胰蛋白酶消化进展的影响而开发的。在最佳切割和分离条件下，成功地实现了在线蛋白水解的精确温度控制和优异的重现性。
