TY - JOUR T1 - Zirconium(IV)-IMAC for phosphopeptide enrichment in phosphoproteomics JF - bioRxiv DO - 10.1101/2020.04.13.038810 SP - 2020.04.13.038810 AU - Ignacio Arribas Diez AU - Ireshyn Govender AU - Previn Naicker AU - Stoyan Stoychev AU - Justin Jordaan AU - Ole N. Jensen Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/04/13/2020.04.13.038810.abstract N2 - 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 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.Competing Interest StatementStoychev S. and Jordaan J. are employees of ReSyn Biosciences and have financial interests in the company. Govender I., and Naicker P., work on collaborative projects across CSIR and ReSyn Biosciences.AcAAcetic acidMeCNAcetonitrileABCAmmonium bicarbonateDDAData-dependent adquisitionFAFormic acidGAGlycolic acidHCDHigher-energy collisional dissociationIMACImmobilized Metal ion Affinity ChromatographyIAAIodoacetamidemgfMascot Generic FileMOACMetal Oxide Affinity Chromatographyp-pepPhosphopeptidesSDCSodium deoxycholateS1/S2/S3/S4/S5Solvent 1/2/3/4/5TATartaric acidTiTitaniumTiO2Titanium oxideTCEPTris(2-carboxyethyl)phosphine hydrochlorideZrZirconiumCAA2-Chloroacetamide ER -