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

Abstract

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 TiO2 magnetic microparticles for phosphopeptide enrichment from simple and complex protein samples prior to phosphopeptide sequencing and characterization by mass spectrometry (LC-MS/MS). We optimized sample-loading conditions to increase phosphopeptide recovery for Zr-IMAC, Ti-IMAC and TiO2 based workflows by 22%, 24% and 35% respectively. The optimized protocol resulted in improved performance of Zr-IMAC over Ti-IMAC and TiO2 as well as HPLC-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.

Current Methods of Post-Translational Modification Analysis and Their Applications in Blood Cancers

“…A recent study demonstrated the improved performance of Zr-IMAC, Ti-IMAC, and TiO2-MOAC following optimization of the binding solvent used during the enrichment protocol. Following optimization, a comparison of the number of phosphopeptides identified between these techniques, as well as the most commonly used Fe-IMAC, revealed that microparticle-based Zr-IMAC enrichment was superior resulting in a higher phosphoproteome coverage [75]. This study also reinforced the widely documented view that combining enrichment techniques results in more robust PTM-focused MS-based analyses.…”

A Rapid and Universal Workflow for Label‐Free‐Quantitation‐Based Proteomic and Phosphoproteomic Studies in Cereals

“…These materials are commercially available and immobilized on diverse supporting matrices for different ways of handling, such as in an agarose resin spin‐down column from Thermo Scientific ™ Pierce ™ , the TiO 2 resin column from Titansphere ® , or in the form of magnetic beads from Cytiva. These different phosphopeptide enrichment approaches result in different phosphopeptides identified (Arribas Diez et al., 2021; Gates et al., 2010; Liang et al., 2007), suggesting that different matrices enrich slightly different types of phosphopeptides. New supporting matrices are continuously being developed (Kupcik et al., 2019).…”

Advances in proteomics sample preparation and enrichment for phosphorylation and glycosylation analysis

“…To date, several emerging methods based on different principles have been developed for PTMs protein sample preparation and enrichment. For example, the enrichment strategies operated in phosphoproteomic studies are molecularly imprinted technology (MIP) [25, 26], ion exchange chromatography (IEC) [27–29], affinity chromatography [30–32], and chemical derivatization [33, 34]. Different from phosphorylation modifications with fixed structures, the diversity of polysaccharides and the interference of glycans on the peptide backbone make the identification of glycopeptides and the study of protein glycosylation encounter more obstacles.…”