Fe3+-NTA magnetic beads as an alternative to spin column-based phosphopeptide enrichment

Protein phosphorylation is a central mechanism of cellular signal transduction in living organisms. Phosphoproteomic studies aim to systematically catalogue, characterize, and comprehend alterations in phosphorylation states across multiple cellular conditions and are often incorporated into global proteomics workflows. Previously, we found that spin column-based Fe3+-NTA enrichment integrated well with our workflow but it remained a bottleneck for methods that require higher throughput or a scale that is beyond the maximum capacity of these columns. Here, we compare our well-established spin column-based enrichment strategy with one encompassing magnetic beads. Our data show little difference in both the number and properties of the phosphopeptides identified when using either method. In all, we illustrate how scalable and automation-friendly magnetic Fe3+-NTA beads can seamlessly substitute spin column-based Fe3+-NTA for global phosphoproteome profiling.