Scalable, Non-denaturing Purification of Phosphoproteins Using Ga3+-IMAC: N2A and M1M2 Titin Components as Study case

  • Michael Adams
  • Jennifer R. Fleming
  • Eva Riehle
  • Tiankun Zhou
  • Thomas Zacharchenko
  • Marija Markovic
  • Olga MayansEmail author


The purification of phosphorylated proteins in a folded state and in large enough quantity for biochemical or biophysical analysis remains a challenging task. Here, we develop a new implementation of the method of gallium immobilized metal chromatography (Ga3+-IMAC) as to permit the selective enrichment of phosphoproteins in the milligram scale and under native conditions using automated FPLC instrumentation. We apply this method to the purification of the UN2A and M1M2 components of the muscle protein titin upon being monophosphorylated in vitro by cAMP-dependent protein kinase (PKA). We found that UN2A is phosphorylated by PKA at its C-terminus in residue S9578 and M1M2 is phosphorylated in its interdomain linker sequence at position T32607. We demonstrate that the Ga3+-IMAC method is efficient, economical and suitable for implementation in automated purification pipelines for recombinant proteins. The procedure can be applied both to the selective enrichment and to the removal of phosphoproteins from biochemical samples.


Phosphorylation FPLC protein purification Titin PKA 



Mass spectrometry


Immobilized metal affinity chromatography


Metal oxide affinity chromatography


Fast protein liquid chromatography


Iminodiacetic acid


Isopropyl β-D-1-thiogalactopyranoside


Tris(2-carboxyethyl)phosphine hydrochloride


cAMP-dependent protein kinase.



We thank the Proteomics Unit of the University of Konstanz for the contribution of mass spectrometry to this work.

Author Contributions

MA, JRF and OM conceived the study; MA and TZa performed experiments and analysed data for M1M2; MA, ER and TZh performed experiments and analysed data for UN2A; MM designed protocols for Ga3+-IMAC column reuse and regeneration; OM, MA and JRF wrote the manuscript; all authors made manuscript revisions.


We acknowledge the financial support of DFG SFB969 and the Leducq Foundation (TNE- 13CVD04). JRF is supported by an EU Marie Sklodowska-Curie Individual Fellowship (TTNPred, 753054).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Human and Animal Participants

This article does not contain any work with human participants or animals performed by any of the authors.


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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiologyUniversity of KonstanzKonstanzGermany
  2. 2.School of BiologyUniversity of LeedsLeedsUK

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