Measurement Solution Research Center, National Institute of Advanced Industrial Science and Technology, Tosu, Saga 841-0052, Japan.
Anal Biochem. 2010 Dec 1;407(1):12-8. doi: 10.1016/j.ab.2010.07.026. Epub 2010 Jul 29.
Proteolysis by sequence-specific proteases is the key step for positive sequencing in proteomic studies integrated with mass spectrometry (MS). The conventional method of in-solution digestion of protein is a time-consuming procedure and has limited sensitivity. In this study, we report a simple and rapid system for the analysis of protein sequence and protein posttranslational modification by multienzymatic reaction in a continuous flow using the enzyme (trypsin, chymotrypsin, or alkaline phosphatase)-immobilized microreactor. The feasibility and performance of the single microreactor and tandem microreactors that were connected by the different microreactors were determined by the digestion of nonphosphoprotein (cytochrome c) and phosphoproteins (β-casein and pepsin A). The single microreactor showed rapid digestion compared with that of in-solution digestions. Multiple digestion by the tandem microreactors showed higher sequence coverage compared with that by in-solution or the single microreactor. Moreover, the tandem microreactor that was made by using the combination of protease-immobilized microreactor and phosphatase-immobilized microreactor showed the capability for phosphorylation site analysis in phosphoproteins without the use of any enrichment strategies or radioisotope labeling techniques. This approach provides a strategy that can be applied to various types of linking microreactor-based multienzymatic reaction systems for proteomic analysis.
序列特异性蛋白酶的蛋白水解是与质谱（MS）集成的蛋白质组学研究中正向测序的关键步骤。蛋白质溶液消化的传统方法是一个耗时的过程，并且灵敏度有限。在这项研究中，我们报告了一种简单而快速的系统，用于通过使用（胰蛋白酶、糜蛋白酶或碱性磷酸酶）固定化微反应器中的多酶反应在连续流中分析蛋白质序列和蛋白质翻译后修饰。通过非磷酸化蛋白（细胞色素 c）和磷酸化蛋白（β-酪蛋白和胃蛋白酶 A）的消化，确定了单微反应器和通过不同微反应器连接的串联微反应器的可行性和性能。与溶液消化相比，单微反应器显示出快速消化。串联微反应器的多次消化显示出比溶液消化或单微反应器更高的序列覆盖率。此外，由蛋白酶固定化微反应器和磷酸酶固定化微反应器组合制成的串联微反应器具有在不使用任何富集策略或放射性同位素标记技术的情况下分析磷酸化蛋白中磷酸化位点的能力。这种方法为基于连接微反应器的多酶反应系统的各种类型的蛋白质组学分析提供了一种策略。
