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

The advent of high-throughput proteomics has created an urgent need for parallel increases in sample processing productivity. However, workflows—particularly those involving PTM profiling—remain highly complex and consist of multiple steps that demand exceptional reproducibility. Many laboratories are transitioning to liquid-handling systems to automate parts of the workflow, but the transition from manual pipetting to full automation of phosphoproteomics can be technically demanding and cost-prohibitive. Here, we describe a practical intermediate solution: the use of a manually operated 96-channel pipettor (Platemaster P220) in combination with a 96-well magnetic pin device (MagPin, VP Scientific). Using these devices, we demonstrate highly efficient and reproducible execution of a complete phosphoproteomics workflow in 96-well format, involving PAC digestion, desalting, phosphoenrichment and second round of desalting within just two days, with low operator workload, minimal sample preparation variability and virtually no risk of error. As part of the workflow, we also present a methodology for producing affordable, highly reproducible Oasis HLB 96-well SPE plates by directly packing HLB sorbent material into tapered filter plates. We comprehensively characterise these custom Oasis HLB plates with respect to loading capacity, acetonitrile percentage required for peptide elution, lipid removal efficiency, and suitability for high-pH fractionation.