Zirconium(IV)-IMAC for phosphopeptide enrichment in phosphoproteomics

Abstract

Phosphopeptide enrichment is an essential step in large-scale, quantitative phosphoproteomics studies 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 phosphopeptide sequencing and characterization by mass spectrometry (LC-MS/MS). We optimized sampleloading conditions to increase phosphopeptide recovery for Zr-IMAC, Ti-IMAC and TiO2 based workflows. The performance of Zr-IMAC was enhanced by 19-22% to recover up to 5173 phosphopeptides from 200 µg of protein extract from HepG2/C3A cells, making Zr-IMAC the preferred method for phosphopeptide enrichment in this study. Ti-IMAC and TiO2 performance were also optimized to improve phosphopeptide numbers by 28% and 35%, respectively. Furthermore, Zr-IMAC based phosphoproteomics in the magnetic microsphere format identified 23% more phosphopeptides than HPLC-based Fe(III)-IMAC for same sample amount (200 µg), thereby adding 37% more uniquely identified phosphopeptides. We conclude that Zr-IMAC improves phosphoproteome coverage and recommend that this affinity enrichment method should be more widely used in biological and biomedical studies of cell signalling and in the search for disease-biomarkers.

Mass Spectrometry-based Proteomics and Glycoproteomics in COVID-19 Biomarkers Identification: A Mini-review

“…IMAC consists of transition metal cations (e.g., Fe 3+ , Ga 3+ , Ti 4+ , Zr 4+ ) chelated to an immobilized substrate, whereas MOAC is a mix of transition metals in a metal oxide matrix (e.g., TiO x ) [ 141 , 142 ]. Both techniques exploit the affinity of deprotonated carboxyl groups to achieve enrichment and are particularly effective in enriching sialylated glycopeptides [ 140 – 143 ]. Hydrophilic interaction chromatography (HILIC) is another important tool for glycoprotein enrichment and characterization and can be applied to a wide range of biomass, such as biological fluids, cancer systems, pathogens, and plants [ 144 – 147 ].…”

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

“…They compared this enrichment to Ti 4+ -IMAC and TiOx enrichments to show this selectivity was specific to their Fe 3+ -IMAC conditions, indicating there is still much to explore as to why some conditions promote co-enrichment of sialylated glycopeptides while others seem more selective for phosphorylated species. As IMAC and MOAC matrices with different metal ions are developed and applied to phosphoproteome characterization (191), it will be important to characterize which translate best for characterization of various subpopulations of the glycoproteome. Additionally, most IMAC and MOAC enrichment studies have focused on by guest on October 30, 2020 N-glycopeptides, so future studies will need to explore its utility for enriching sialylated, sulfated, and otherwise anionic O-glycopeptides, presumably following enzymatic removal of confounding N-glycans.…”