High-Throughput Proteomics Sample Preparation Using a 96-Channel Pipettor and Magnetic Pin Device

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• High-throughput proteomics, especially post-translational modification (PTM) profiling, faces challenges in automation, reproducibility, and cost.
• Current automated workflows are often complex and expensive, limiting accessibility for many research labs.

Purpose of the Study:

• To present a practical, cost-effective intermediate solution for reproducible phosphoproteomics using manually operated 96-channel devices.
• To simplify and enhance the efficiency of sample processing in high-throughput proteomics.
• To serve as a stepping stone towards full automation of proteomics workflows.

Main Methods:

• Utilized a Gilson Platemaster P220 pipettor and VP Scientific 96-well magnetic pin device for manual operation.
• Developed a cost-efficient method for generating 96-well solid-phase extraction plates using Oasis HLB sorbent.
• Optimized protein aggregation capture (PAC/SP3) digestion by demonstrating that continuous bead suspension is not required.

Main Results:

• Achieved robust and reproducible phosphoproteomics in a 96-well format within 2 days.
• Successfully completed protein aggregation capture (PAC/SP3) digestion, desalting, phosphopeptide enrichment, and a second desalting step.
• Characterized the performance of custom-packed 96-well solid-phase extraction plates for loading capacity, lipid removal, and fractionation suitability.
• Demonstrated simplified PAC digestion by brief aspiration, eliminating the need for orbital shaking.

Conclusions:

• The presented manual 96-channel workflow offers a practical and reproducible approach to phosphoproteomics.
• Innovations in solid-phase extraction plate generation and digestion methods reduce cost and complexity.
• This workflow familiarizes users with 96-channel devices, facilitating a transition towards fully automated high-throughput proteomics.