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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
Article
  • 27 Downloads

Abstract

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.

Keywords

Phosphorylation FPLC protein purification Titin PKA 

Abbreviations

MS

Mass spectrometry

IMAC

Immobilized metal affinity chromatography

MOAC

Metal oxide affinity chromatography

FPLC

Fast protein liquid chromatography

IDA

Iminodiacetic acid

IPTG

Isopropyl β-D-1-thiogalactopyranoside

TCEP

Tris(2-carboxyethyl)phosphine hydrochloride

PKAcα

cAMP-dependent protein kinase.

Notes

Acknowledgements

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.

Funding

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.

References

  1. 1.
    Humphrey SJ, James DE, Mann M (2015) Protein phosphorylation: a major switch mechanism for metabolic regulation. Trends Endocrinol Metab 26:676–687CrossRefGoogle Scholar
  2. 2.
    Dephoure N, Gould KL, Gygi SP, Kellogg DR (2013) Mapping and analysis of phosphorylation sites: a quick guide for cell biologists. Mol Biol Cell 24:535–542CrossRefGoogle Scholar
  3. 3.
    Fíla J, Honys D (2012) Enrichment techniques employed in phosphoproteomics. Amino Acids 43:1025–1047CrossRefGoogle Scholar
  4. 4.
    Li Y, Xu X, Qi D, Deng C, Yang P, Zhang X (2008) Novel Fe3O4@TiO2 core-shell microspheres for selective enrichment of phosphopeptides in phosphoproteome analysis. J Proteome Res 7:2526–2538CrossRefGoogle Scholar
  5. 5.
    Lin H, Deng C (2016) Development of immobilized Sn(4+) affinity chromatography material for highly selective enrichment of phosphopeptides. Proteomics 16:2733–2741CrossRefGoogle Scholar
  6. 6.
    Zou X, Jie J, Yang B (2017) Single-step enrichment of N-glycopeptides and phosphopeptides with novel multifunctional Ti(4+)-immobilized dendritic polyglycerol coated chitosan nanomaterials. Anal Chem 89:7520–7526CrossRefGoogle Scholar
  7. 7.
    Yang DS, Ding XY, Min HP, Li B, Su MX, Niu MM, Di B, Yan F (2017) Design and synthesis of an immobilized metal affinity chromatography and metal oxide affinity chromatography hybrid material for improved phosphopeptide enrichment. J Chromatogr A 1505:56–62CrossRefGoogle Scholar
  8. 8.
    Posewitz MC, Tempst P (1999) Immobilized gallium(III) affinity chromatography of phosphopeptides. Anal Chem 71:2883–2892CrossRefGoogle Scholar
  9. 9.
    Machida M, Kosako H, Shirakabe K, Kobayashi M, Ushiyama M, Inagawa J, Hirano J, Nakano T, Bando Y, Nishida E, Hattori S (2007) Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis. FEBS J 274:1576–1587CrossRefGoogle Scholar
  10. 10.
    Lai AC, Tsai CF, Hsu CC, Sun YN, Chen YJ (2012) Complementary Fe(3+)- and Ti(4+)-immobilized metal ion affinity chromatography for purification of acidic and basic phosphopeptides. Rapid Commun Mass Spectrom 26:2186–2194CrossRefGoogle Scholar
  11. 11.
    Zhu L, Zhang J, Guo Y (2014) Enhanced detection and desalting free protocol for phosphopeptides eluted from immobilized Fe (III) affinity chromatography in direct MALDI TOF analysis. J Proteomics 96:360–365CrossRefGoogle Scholar
  12. 12.
    Yao Y, Dong J, Dong M, Liu F, Wang Y, Mao J, Ye M, Zou H (2017) An immobilized titanium (IV) ion affinity chromatography adsorbent for solid phase extraction of phosphopeptides for phosphoproteome analysis. J Chromatogr A 1498:22–28CrossRefGoogle Scholar
  13. 13.
    Aryal UK, Ross AR (2010) Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry. Rapid Commun Mass Spectrom 24:219–231CrossRefGoogle Scholar
  14. 14.
    Sykora C, Hoffmann R, Hoffmann P (2007) Enrichment of multiphosphorylated peptides by immobilized metal affinity chromatography using Ga(III)- and Fe(III)-complexes. Protein Pept Lett 14:489–496CrossRefGoogle Scholar
  15. 15.
    Aryal UK, Olson DJ, Ross AR (2008) Optimization of immobilized gallium (III) ion affinity chromatography for selective binding and recovery of phosphopeptides from protein digests. J Biomol Tech 19:296–310Google Scholar
  16. 16.
    Albuquerque CP, Smolka MB, Payne SH, Bafna V, Eng J, Zhou H (2008) A multidimensional chromatography technology for in-depth phosphoproteome analysis. Mol Cell Proteomics 7:1389–1396CrossRefGoogle Scholar
  17. 17.
    Yue XS, Hummon AB (2013) Combination of multistep IMAC enrichment with high-pH reverse phase separation for in-depth phosphoproteomic profiling. J Proteome Res 12:4176–4186CrossRefGoogle Scholar
  18. 18.
    Steen H, Stensballe A, Jensen ON (2007) Phosphopeptide Purification by IMAC with Fe(III) and Ga(III). CSH Protoc  https://doi.org/10.1101/pdb.prot4607 Google Scholar
  19. 19.
    Liu WR, Wang YS, Wan W (2011) Synthesis of proteins with defined posttranslational modifications using the genetic noncanonical amino acid incorporation approach. Mol Biosyst 7:38–47CrossRefGoogle Scholar
  20. 20.
    Ravi A, Guo S, Rasala B, Tran M, Mayfield S, Nikolov ZL (2018) Separation options for phosphorylated osteopontin from transgenic microalgae Chlamydomonas reinhardtii. Int J Mol Sci  https://doi.org/10.3390/ijms19020585 Google Scholar
  21. 21.
    Zhou T, Fleming JR, Franke B, Bogomolovas J, Barsukov I, Rigden DJ, Labeit S, Mayans O (2016) CARP interacts with titin at a unique helical N2A sequence and at the domain Ig81 to form a structured complex. FEBS Lett 590:3098–3110CrossRefGoogle Scholar
  22. 22.
    Kinoshita E, Yamada A, Takeda H, Kinoshita-Kikuta E, Koike T (2005) Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins. J Sep Sci 28:155–162CrossRefGoogle Scholar
  23. 23.
    Johnson WC (1999) Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins 35:307–312CrossRefGoogle Scholar
  24. 24.
    Whitmore L, Wallace BA (2008) Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 89:392–400CrossRefGoogle Scholar
  25. 25.
    Krüger M, Kötter S (2016) Titin, a central mediator for hypertrophic signaling, exercise-induced mechanosignaling and skeletal muscle remodeling. Front Physiol 7:1–8.  https://doi.org/10.3389/fphys.2016.00076 CrossRefGoogle Scholar
  26. 26.
    Blom N, Gammeltoft S, Brunak S (1999) Sequence- and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294:1351–1362CrossRefGoogle Scholar
  27. 27.
    Bang ML1, Centner T, Fornoff F, Geach AJ, Gotthardt M, McNabb M, Witt CC, Labeit D, Gregorio CC, Granzier H, Labeit S (2001) The complete gene sequence of titin, expression of an unusual ≈ 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-line to I-band linking system. Circ Res 89:1065–1072CrossRefGoogle Scholar
  28. 28.
    Miller MK, Bang ML, Witt CC, Labeit D, Trombitas C, Watanabe K, Granzier H, McElhinny AS, Gregorio CC, Labeit S (2003) The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules. J Mol Biol 333:951–964CrossRefGoogle Scholar
  29. 29.
    Tiffany H, Sonkar K, Gage MJ (2017) The insertion sequence of the N2A region of titin exists in an extended structure with helical characteristics. Biochim Biophys Acta Proteins Proteom 1865:1–10CrossRefGoogle Scholar
  30. 30.
    Drozdetskiy A, Cole C, Procter J, Barton GJ (2015) JPred4: A protein secondary structure prediction server. Nucleic Acids Res 43:W389–W394CrossRefGoogle Scholar
  31. 31.
    Kozlowski LP, Bujnicki JM (2012) MetaDisorder: a meta-server for the prediction of intrinsic disorder in proteins. BMC Bioinformatics 13:111CrossRefGoogle Scholar
  32. 32.
    Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinform 9:1–8CrossRefGoogle Scholar
  33. 33.
    Zacharchenko T, von Castelmur E, Rigden DJ, Mayans O (2015) Structural advances on titin: towards an atomic understanding of multi-domain functions in myofilament mechanics and scaffolding. Biochem Soc Trans 43:850–855CrossRefGoogle Scholar
  34. 34.
    Funabara D, Kinoshita S, Watabe S, Siegman MJ, Butler TM, Hartshorne DJ (2001) Phosphorylation of molluscan twitchin by the cAMP-dependent protein kinase. Biochemistry 40:2087–2095CrossRefGoogle Scholar
  35. 35.
    Funabara D, Hamamoto C, Yamamoto K, Inoue A, Ueda M, Osawa R, Kanoh S, Hartshorne DJ, Suzuki S, Watabe S (2007) Unphosphorylated twitchin forms a complex with actin and myosin that may contribute to tension maintenance in catch. J Exp Biol 210:4399–4410CrossRefGoogle Scholar

Copyright information

© 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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