biostudies-literatureArad MNatural Sciences and Engineering Research Council of CanadaUniversity of the Fraser ValleyBC Knowledge Development FundCanada Foundation for InnovationGenome British Columbia18880-18889https://www.ebi.ac.uk/biostudies/studies/S-EPMC11603402biostudies-literatureUnknown96(47)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, <i>in situ</i> 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 efficiencAnalytical chemistryDevelopment of an Automated, Ultra-Rapid Bottom-Up Proteomics Workflow Utilizing Alginate-Based Hydrogels.PMC11603402374PRONestman ZJereb DFordwour OBalagtas RFrey CShi YKu KHare RSidhu AMoon KMGhafourifar GFoster LJArad MfalseDevelopment 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, <i>in situ</i> 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 efficienc2024-01-01T00:00:00Z2024 Nov2026-06-01T16:23:15.824Z2025-04-04T02:33:27.245ZS-EPMC116034023952841510.1021/acs.analchem.4c04846