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

## TL;DR

This paper introduces a cost-effective, semi-automated workflow for high-throughput phosphoproteomics using 96-channel devices, reducing workload and variability.

## Contribution

A practical, semi-automated phosphoproteomics workflow using 96-channel devices with cost-efficient solid-phase extraction plates and simplified digestion.

## Key findings

- A 96-well phosphoproteomics workflow was completed in 2 days with minimal operator workload.
- Custom solid-phase extraction plates using Oasis HLB sorbent were characterized for loading capacity and lipid removal.
- PAC digestion can be simplified by aspirating beads without requiring continuous shaking.

## Abstract

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.