Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 8, 12800 Prague, Czech Republic.
Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Aug 1;1228:123866. doi: 10.1016/j.jchromb.2023.123866. Epub 2023 Aug 25.
Liquid chromatography coupled with mass spectrometry is widely used in the field of proteomic analysis after off-line protein digestion. On-line digestion with chromatographic column connected in a series with immobilized enzymatic reactor is not often used approach. In this work we investigated the impact of chromatographic conditions on the protein digestion efficiency. The investigation of trypsin reactor activity was performed by on-line digestion of N-α-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA), followed by separation of the digests on the mixed-mode column. Two trypsin column reactors with the different trypsin coverage on the bridged ethylene hybrid particles were evaluated. To ensure optimal trypsin activity, the separation temperature was set at 37.0 °C and the pH of the mobile phase buffer was maintained at 8.5. The on-line digestion itself ongoing during the initial state of gradient was carried out at a low flow rate using a mobile phase that was free of organic modifiers. Proteins such as cytochrome C, enolase, and myoglobin were successfully digested on-line without prior reduction or alkylation, and the resulting peptides were separated using a mixed-mode column. Additionally, proteins that contain multiple cysteines, such as α-lactalbumin, albumin, β-lactoglobulin A, and conalbumin, were also successfully digested on-line (after reduction and alkylation). Moreover, trypsin immobilized enzymatic reactors were utilized for over 300 injections without any noticeable loss of digestion activity.
液相色谱-质谱联用技术在离线蛋白质消化后广泛应用于蛋白质组学分析领域。在线与色谱柱串联的固定化酶反应器的消化方法并不常用。本工作研究了色谱条件对蛋白质消化效率的影响。通过在线消化 N-α-苯甲酰-L-精氨酸 4-硝基苯胺盐酸盐（BAPNA），考察了胰蛋白酶反应器的活性，然后在混合模式柱上分离酶解产物。评价了两种具有不同桥接乙烯杂化颗粒覆盖度的胰蛋白酶柱反应器。为确保最佳胰蛋白酶活性，分离温度设定为 37.0°C，流动相缓冲液 pH 值维持在 8.5。在线消化在初始梯度阶段以低流速进行，流动相不含有机改性剂。无需还原或烷基化，即可成功在线消化细胞色素 C、烯醇酶和肌红蛋白等蛋白质，并使用混合模式柱分离得到的肽段。此外，还成功在线消化了含有多个半胱氨酸的蛋白质，如α-乳白蛋白、白蛋白、β-乳球蛋白 A 和伴白蛋白（还原和烷基化后）。此外，固定化酶胰蛋白酶反应器在 300 多次注射后仍保持良好的消化活性，没有明显的活性损失。
