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, <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 efficiency.
Authors
- Osei Boakye Fordwour
- Connor Frey (ORCID: https://orcid.org/0000-0002-4080-3030)
- Kenneth Ku (ORCID: https://orcid.org/0000-0002-8905-5432)
- Rhien Hare (ORCID: https://orcid.org/0000-0002-6732-956X)
- Leonard J. Foster (ORCID: https://orcid.org/0000-0001-8551-4817)
- Golfam Ghafourifar (ORCID: https://orcid.org/0000-0001-8024-1792)
- Arshdeep Sidhu (ORCID: https://orcid.org/0000-0002-2851-1019)
- Yuming Shi (ORCID: https://orcid.org/0009-0008-4027-2373)
- Maor Arad (ORCID: https://orcid.org/0000-0003-4618-9868)
- Kyung‐Mee Moon (ORCID: https://orcid.org/0000-0003-3796-720X)
- Ronald Balagtas (ORCID: https://orcid.org/0009-0009-7728-3751)
- Dario Jereb
- Zach Nestman
Institutions
- University of British Columbia (CA)
- University of the Fraser Valley (CA)
- Simon Fraser University (CA)
- Canada's Michael Smith Genome Sciences Centre (CA)
Publication Details
- Journal
- Analytical Chemistry
- Published
- 2024-11-11
- DOI
- https://doi.org/10.1021/acs.analchem.4c04846
- Citations
- 6
- Primary Topic
- Advanced Proteomics Techniques and Applications
- Type
- article
- Field-Weighted Citation Impact
- 1.44
Funders
- University of the Fraser Valley
- Canada Foundation for Innovation
- Genome British Columbia
- Simon Fraser University
- Natural Sciences and Engineering Research Council of Canada