Zirconium(IV)-IMAC Revisited: Improved Performance and Phosphoproteome Coverage by Magnetic Microparticles for Phosphopeptide Affinity Enrichment

Phosphopeptide enrichment is an essential step in large-scale, quantitative phosphoproteomics by mass spectrometry. Several phosphopeptide affinity enrichment techniques exist, such as immobilized metal-ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC). We compared zirconium(IV) IMAC (Zr-IMAC) magnetic microparticles to more commonly used titanium(IV) IMAC (Ti-IMAC) and TiO 2 magnetic microparticles for phosphopeptide enrichment from simple and complex protein samples prior to phosphopeptide sequencing and characterization by mass spectrometry (liquid chromatography–tandem mass spectrometry, LC–MS/MS). We optimized sample-loading conditions to increase phosphopeptide recovery for Zr-IMAC-, Ti-IMAC-, and TiO 2 -based workflows by 22, 24, and 35%, respectively. The optimized protocol resulted in improved performance of Zr-IMAC over Ti-IMAC and TiO 2 as well as high-performance liquid chromatography-based Fe(III)-IMAC with up to 23% more identified phosphopeptides. The different enrichment chemistries showed a high degree of overlap but also differences in phosphopeptide selectivity and complementarity. We conclude that Zr-IMAC improves phosphoproteome coverage and recommend that this complementary and scalable affinity enrichment method is more widely used in biological and biomedical studies of cell signaling and the search for biomarkers. Data are available via ProteomeXchange with identifier PXD018273.