A new approach to sample preparation and enzymatic digestion in bottom-up proteomics has been developed using alginate-based hydrogel entrapment of enzymes. This hydrogel facilitates rapid and room-temperature digestions with multienzyme capabilities. Three methodologies were tested: within microcentrifuge tubes, in situ pipette tips, and automated robotic liquid handling. Factorial experimental design identified a 1 h, room temperature, pepsin–trypsin dual-enzyme digestion as optimal for sequence coverage and protein group identification, comparable to a gold-standard overnight proteomic protocol. This method promises significant advancements in proteomic analysis by enhancing reusability, speed, throughput, convenience, and cost-effectiveness, without hindering digestion efficiency.

中文翻译：

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利用藻酸盐基水凝胶开发自动化、超快速、自下而上的蛋白质组学工作流程


在自下而上的蛋白质组学中，已经开发了一种使用基于藻酸盐的水凝胶包埋酶的样品制备和酶消化的新方法。这种水凝胶具有多酶功能，有助于在室温下进行快速消化。测试了三种方法：微量离心管内、原位移液器吸头和自动机器人液体处理。析因实验设计确定 1 小时、室温、胃蛋白酶-胰蛋白酶双酶消化是序列覆盖和蛋白质组鉴定的最佳方法，与金标准过夜蛋白质组学方案相当。该方法通过提高可重用性、速度、通量、便利性和成本效益，而不影响消化效率，有望在蛋白质组学分析方面取得重大进展。