Zwitter-ionic monolith-based spintip column coupled with Evosep One liquid chromatography for high-throughput...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biotechnology

Background:

• High-throughput proteomics requires sensitive and reproducible workflows for large clinical cohorts.
• Existing methods often face challenges with sample input volume and processing time.

Purpose of the Study:

• To develop and validate a streamlined proteomic workflow for large-scale clinical sample analysis.
• To integrate zwitter-ionic monolith-based spintip (ZIM-Tip) technology with the Evosep One liquid chromatography system.

Main Methods:

• Zwitter-ionic monolith-based spintip (ZIM-Tip) preparation via photo-initiated free radical polymerization.
• On-tip sample processing including protein concentration, reduction, alkylation, and enzymatic digestion (2 hours).
• Peptide analysis using Evosep One LC-MS/MS on a timsTOF Pro mass spectrometer in data-dependent acquisition mode.

Main Results:

• Consistent identification of over 4000 proteins from 1 µg of cell lysate per 1-hour LC-MS/MS run.
• Processing capacity of at least 20 samples per day with low sample loss (~10%).
• Excellent quantification precision (median CV < 20% intra-day, < 30% inter-day) and high sample-to-sample correlation (average Pearson R > 0.90].

Conclusions:

• The ZIM-Tip and Evosep One integrated workflow provides a robust solution for high-throughput clinical proteomics.
• This technology effectively handles large sample cohorts with low sample input requirements.
• The workflow ensures in-depth proteome coverage and high reproducibility, crucial for clinical research.