Zirconium(IV)-IMAC for phosphopeptide enrichment in phosphoproteomics

ABSTRACTPhosphopeptide 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 sample-loading 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.

Preparation of Nano-TiO2 Modified Temperature-Responsive Chromatographic Materials for Enrichment of Phosphopeptides

Chromatographic stationary phases with specific capturing phosphoproteins is widely used in biological sample pretreatment. However, when captured protein is released, it is required to change the pH of the mobile phase or to use an eluent. Usually, the mobile phase or eluent are salt solutions with high concentration and extreme pH or toxic organic reagents. In this situation, these reagents will destroy the activity and structure of phosphorylated proteins. In addition, the mobile phase after switching the column takes longer time to restore the balance, reducing the experimental efficiency. In order to solve the these problems, we introduce temperature-reponsive materials into the chromatographic stationary phase to achieve the capture and release of phosphorylated proteins by changing the temperature only, in which we use water as the mobile phase. This approach overcomes the drawbacks of traditional methods, and makes the separation process safe and simple. Based on the surface initiated Reversible Addition Fragmentation Chain Transfer Polymerization (SI-RAFT) method, silica@pNIPAAm-nanoTiO2, a kind of Metal Oxide Affinity Chromatography, was synthesized by the rapid introduction of functional groups. The synthesis of silica@pNIPAAm-nanoTiO2was confirmed by infrared and X-ray photoelectron spectroscopy. The grafting rate and the lowest critical temperature were measured by TG and DSC. The results showed that the material had qualified temperature-sensitive properties. The grafting conformation and mobile phase pH of the material were optimized before testing the properties and found that when the material grafting ratio was 10% -15%, the graft density was 30%, and the mobile phase pH was 6, it had the best separate effect. Finally, the material successfully achieved the capture and release of adenosine triphosphate and casein phosphopeptides.