Department of Chemical Engineering and Analytical Chemistry, Institute for Research on Nutrition and Food Safety (INSA·UB), University of Barcelona, 1-11 Marti i Franques street, 3rd floor, Barcelona 08028, Spain.
Department of Chemical Engineering and Analytical Chemistry, Institute for Research on Nutrition and Food Safety (INSA·UB), University of Barcelona, 1-11 Marti i Franques street, 3rd floor, Barcelona 08028, Spain.
Talanta. 2019 Jul 1;199:116-123. doi: 10.1016/j.talanta.2019.02.039. Epub 2019 Feb 10.
In this study, we present the use of microreactors packed with immobilized trypsin particles for the rapid and efficient bottom-up analysis of proteins by on-line immobilized enzyme microreactor capillary electrophoresis mass spectrometry (IMER-CE-MS). The results obtained digesting β-lactoglogulin (β-LG) off-line with free trypsin in solution and with immobilized trypsin particles were taken as a reference for the optimization of the on-line protein digestion. Under the optimized conditions, on-line digestion, separation and characterization of the protein digests were possible in less than 30 min. The limit of detection for complete sequence coverage was around 10 µg mL (~500 µM) of β-LG, the repeatability was comparable to the off-line digestion methods and the microreactor could be reused until thirty times. The good performance of IMER-CE-MS was also demonstrated for several other proteins as α-casein (α-CSN), β-casein (β-CSN), and κ-casein (κ-CSN), as well as for a complex protein mixture (an Escherichia coli whole cell lysate).
在本研究中，我们展示了使用填充固定化胰蛋白酶颗粒的微反应器，通过在线固定化酶微反应器毛细管电泳质谱联用（IMER-CE-MS）快速有效地进行蛋白质的从头分析。通过在线消化β-乳球蛋白（β-LG），并与游离胰蛋白酶溶液和固定化胰蛋白酶颗粒离线消化的结果进行了比较，以优化在线蛋白质消化。在优化的条件下，可在不到 30 分钟的时间内完成蛋白质消化物的在线分离和鉴定。完整序列覆盖的检测限约为 10μg/mL（~500μM）β-LG，重复性与离线消化方法相当，微反应器可重复使用三十次以上。IMER-CE-MS 对其他几种蛋白质，如α-酪蛋白（α-CSN）、β-酪蛋白（β-CSN）和κ-酪蛋白（κ-CSN），以及复杂的蛋白质混合物（大肠杆菌全细胞裂解物），也表现出良好的性能。
