Proteomics pp 47-60 | Cite as

Optimized Enrichment of Phosphoproteomes by Fe-IMAC Column Chromatography

  • Benjamin Ruprecht
  • Heiner Koch
  • Petra Domasinska
  • Martin Frejno
  • Bernhard KusterEmail author
  • Simone Lemeer
Part of the Methods in Molecular Biology book series (MIMB, volume 1550)


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 TiO2 or 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).

Key words

Phosphorylation Proteomics Phosphocapture LC-MS 





Acquisition gain control






Formic acid


Fetal calf serum


Higher energy collision induced dissociation




High-performance liquid chromatography


Inner diameter


Immobilized metal ion affinity chromatography




Mass spectrometry


Tandem mass spectrometry


Phosphate buffered saline


parts per million


Peptide spectrum match


Phosphotyrosine, -serine, -threonine


Trifluoro acetic acid


Titanium dioxide








Zirconium dioxide


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Benjamin Ruprecht
    • 1
    • 2
  • Heiner Koch
    • 1
    • 3
    • 4
  • Petra Domasinska
    • 5
    • 6
  • Martin Frejno
    • 1
    • 7
  • Bernhard Kuster
    • 1
    • 2
    • 3
    • 4
    • 8
    Email author
  • Simone Lemeer
    • 1
    • 2
    • 9
  1. 1.Chair of Proteomics and Bioanalytics, Technische Universität MünchenFreisingGermany
  2. 2.Center for Integrated Protein Science Munich (CIPSM)FreisingGermany
  3. 3.German Cancer Consortium (DKTK)HeidelbergGermany
  4. 4.German Cancer Research Center (DKFZ)HeidelbergGermany
  5. 5.Biomedical Research CenterUniversity Hospital Hradec KraloveHradec KraloveCzech Republic
  6. 6.Faculty of Chemical Technology, Department of Biological and Biochemical SciencesUniversity of PardubicePardubiceCzech Republic
  7. 7.Department of OncologyUniversity of OxfordOxfordUK
  8. 8.Bavarian Biomolecular Mass Spectrometry CenterTechnische Universität MünchenFreisingGermany
  9. 9.Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical SciencesUtrecht UniversityUtrechtThe Netherlands

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