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Optimizing Proximity Proteomics on the EvoSep-timsTOF LC-MS System
Julia Kitaygorodsky1,2, Brendon Seale1, Vesal Kasmaeifar1,2
1Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Sinai Health, Toronto, Ontario, Canada.
Proteomics
|July 11, 2025
Summary
Optimizing proximity-dependent biotinylation (BioID) with EvoSep-timsTOF mass spectrometry increases throughput 15-fold and identifies more proteins. This new workflow minimizes peptide carryover, eliminating wash steps and enabling 60 samples daily.
Area of Science:
- Proteomics
- Biochemistry
- Cell Biology
Background:
- Proximity-dependent biotinylation (BioID) identifies protein interactors by labeling proximal proteins with biotin.
- Peptide carryover in mass spectrometry (MS) limits BioID throughput due to necessary wash cycles.
- Existing BioID workflows require lengthy intersample washes, reducing sample processing efficiency.
Purpose of the Study:
- To optimize BioID sample acquisition using an EvoSep LC system coupled to a timsTOF mass spectrometer.
- To increase throughput and sensitivity while reducing peptide carryover in BioID-MS workflows.
- To systematically evaluate the EvoSep-timsTOF system for BioID and compare it to established methods.
Main Methods:
- Implemented an EvoSep LC system with a timsTOF mass spectrometer for BioID sample analysis.
- Optimized sample loading and gradient settings for the EvoSep-timsTOF platform.
- Compared protein identification and carryover levels against a previously standardized BioID workflow.
Main Results:
- Achieved an approximately 15-fold increase in throughput, processing 60 samples per day.
- Identified nearly double the number of proteins compared to the previous workflow with enhanced sensitivity.
- Demonstrated extremely limited peptide carryover, even without intersample washing, allowing for elimination of wash cycles.
Conclusions:
- The EvoSep-timsTOF system significantly enhances BioID throughput and sensitivity.
- Eliminating intersample washing is feasible without compromising the identification of proximal interactors.
- Optimized BioID workflows using advanced instrumentation improve the efficiency and scope of proteomic studies.
