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 systematically catalogue and characterize alterations in phosphorylation states across multiple cellular conditions and are often incorporated into global proteomics experiments. Previously, we found that spin column-based Fe3+-NTA enrichment integrated well with our workflow but remained a bottleneck for methods that require higher throughput or a scale that is beyond the 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 when using either method in terms of the number of identified phosphopeptides as well as their physicochemical properties. In all, we illustrate how the potentially scalable and automation-friendly magnetic Fe3+-NTA beads can seamlessly substitute spin column-based Fe3+-NTA agarose beads for global phosphoproteome profiling. SIGNIFICANCE: Protein phosphorylation plays a key role in regulating a multitude of biological processes and can lead to insights into disease pathogenesis. Methodologies which can efficiently enrich phosphopeptides in a scalable and high-throughput manner are essential for profiling dynamic phosphoproteomes. Here we compare two phosphopeptide enrichment workflows, a well-established spin column-based strategy with agarose Fe3+-NTA beads and a strategy using magnetic Fe3+-NTA beads. Our data suggest that the scalable and automation-friendly magnetic bead-based workflow is an equivalent, but more flexible, enrichment strategy for phosphoproteome profiling experiments.