Phosphorylation is among the most important post-translational modifications of proteins and has numerous regulatory functions across all domains of life. However, phosphorylation is often substoichiometric, requiring selective and sensitive methods to enrich phosphorylated peptides from complex cellular digests. Various methods have been devised for this purpose and we have recently described a Fe-IMAC HPLC column chromatography setup which is capable of comprehensive, reproducible, and selective enrichment of phosphopeptides out of complex peptide mixtures. In contrast to other formats such as StageTips or batch incubations using TiO2or Ti-IMAC beads, Fe-IMAC HPLC columns do not suffer from issues regarding incomplete phosphopeptide binding or elution and enrichment efficiency scales linearly with the amount of starting material. Here, we provide a step-by-step protocol for the entire phosphopeptide enrichment procedure including sample preparation (lysis, digestion, desalting), Fe-IMAC column chromatography (column setup, operation, charging), measurement by LC-MS/MS (nHPLC gradient, MS parameters) and data analysis (MaxQuant). To increase throughput, we have optimized several key steps such as the gradient time of the Fe-IMAC separation (15 min per enrichment), the number of consecutive enrichments possible between two chargings (>20) and the column recharging itself (<1 h). We show that the application of this protocol enables the selective (>90 %) identification of more than 10,000 unique phosphopeptides from 1 mg of HeLa digest within 2 h of measurement time (Q Exactive Plus).

Biomedical Research Center University Hospital Hradec Kralove Hradec Kralove Czech Republic Faculty of Chemical Technology Department of Biological and Biochemical Sciences University of Pardubice Pardubice Czech Republic

Chair of Proteomics and Bioanalytics Technische Universität München Emil Erlenmeyer Forum 5 85354 Freising Germany Center for Integrated Protein Science Munich Freising Germany

Chair of Proteomics and Bioanalytics Technische Universität München Emil Erlenmeyer Forum 5 85354 Freising Germany Center for Integrated Protein Science Munich Freising Germany Biomolecular Mass Spectrometry and Proteomics Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences Utrecht University Utrecht The Netherlands

Chair of Proteomics and Bioanalytics Technische Universität München Emil Erlenmeyer Forum 5 85354 Freising Germany Center for Integrated Protein Science Munich Heidelberg Germany Bavarian Biomolecular Mass Spectrometry Center Technische Universität München Freising Germany

Chair of Proteomics and Bioanalytics Technische Universität München Emil Erlenmeyer Forum 5 85354 Freising Germany Department of Oncology University of Oxford Oxford UK

Chair of Proteomics and Bioanalytics Technische Universität München Emil Erlenmeyer Forum 5 85354 Freising Germany German Cancer Consortium Heidelberg Germany

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