Development of an Automated, Ultra-Rapid Bottom-Up Proteomics Workflow Utilizing Alginate-Based Hydrogels.

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.