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

High-throughput proteomics requires efficient and highly reproducible sample processing, yet workflows─particularly for PTM profiling─remain complex and costly to fully automate. Here, we present a practical intermediate solution using manually operated 96-channel devices: the Gilson Platemaster P220 pipettor and VP Scientific 96-well magnetic pin device. Using this setup, we achieved robust and reproducible phosphoproteomics in a 96-well format, completing protein aggregation capture (PAC/SP3) digestion, desalting, phosphopeptide enrichment, and a second desalting step within 2 days while minimizing operator workload and variability. Several innovations enable this workflow. First, we describe a cost-efficient method to generate 96-well solid-phase extraction plates by directly packing the Oasis HLB sorbent into tapered filter plates. We extensively characterize these plates in terms of loading capacity, lipid removal efficiency, and suitability for high-pH fractionation. Second, we demonstrate that efficient PAC digestion does not require continuous bead suspension; instead, digestion can be achieved by briefly aspirating beads in protease solution, eliminating the need for orbital shaking and simplifying automation. The presented workflow familiarizes users with 96-channel devices and hence serves as a good step toward full automation.