Advertisement

Molecular Biotechnology

, Volume 33, Issue 3, pp 179–190 | Cite as

Combinations of SPR and MS for characterization of native and recombinant proteins in cell lysates

  • Jonas Borch
  • Peter Roepstorff
Research

Abstract

Surface plasmon resonance and mass spectrometry (SPR-MS) has been combined for quality check of recombinant 6xHis-tagged 14-3-3 proteins expressed in Escherichia coli. Lysates were injected over an SPR sensorchip with immobilized Ni2+ for SPR analysis of the specific Ni2+ binding response and stability. To validate the identity, intactness and homogeneity of the captured proteins were eluted for mass spectrometric analysis of intact molecular weight and peptide mass mapping. Additionally, the captured recombinant proteins were investigated for specific binding to known phosphorylated ligands of 14-3-3 proteins in order to test their activity.

Specific binding of recombinant and native 14-3-3 proteins in complex mixtures to immobilized phosphopeptides and subsequent elution was also tested by SPR-MS. Ammonium sulfate precipitate fractions from lysates of E. coli expressing 14-3-3 protein and of cauliflower were investigated for specific binding to the phosphopeptide ligands immobilized on a sensorchip by SPR. Subsequently, the bound protein was eluted and analyzed by MS for characterization of intact mass and peptide mass mapping.

Index Entries

Surface plasmon resonance/mass spectrometry (SPR/MS) 14-3-3 proteins affinity purification recombinant 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Borman, S. (1987) Analytical biotechnology of recombinant products. Anal. Chem. 59, 969A-973A.PubMedGoogle Scholar
  2. 2.
    Fägerstam, L.G. (1991) A non-label technology for real-time biospecific interaction analysis. Tech. Prot. Chem. II, 65–71.Google Scholar
  3. 3.
    Mattei, B., Borch, J., and Roepstorff, P. (2004) Biomolecular interaction analysis and MS. Anal. Chem. 76, 18A-25A.CrossRefGoogle Scholar
  4. 4.
    Buijs, J. and Franklin, G.C. (2005) SPR-MS in functional proteomics. Brief Funct. Genomic Proteomic.Google Scholar
  5. 5.
    Zhukov, A., Schurenberg, M., Jansson, O., Areskoug, D., and Buijs, J. (2004) Integration of surface plasmon resonance with mass spectrometry: automated ligand fishing and sample preparation for MALDI MS using a Biacore 3000 biosensor. J. Biomol. Tech. 15, 112–119.PubMedGoogle Scholar
  6. 6.
    Nedelkov, D. and Nelson, R.W. (2003) Design and use of multi-affinity surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS): a step toward the design of SPR/MS arrays. J. Mol. Recognit. 16, 15–19.PubMedCrossRefGoogle Scholar
  7. 7.
    Ferl, R.J. (2004) 14-3-3 proteins: regulation of signal-induced events. Physiol Plantarum. 120, 173–178.PubMedCrossRefGoogle Scholar
  8. 8.
    Muslin, A.J. and H.M. Xing. (2000) 14-3-3 proteins: regulation of subcellular localization by molecular interference. Cell. Signal. 12, 703–709.PubMedCrossRefGoogle Scholar
  9. 9.
    Rosenquist, M., Sehnke, P., Ferl, R. J., Sommarin, M., and Larsson, C. (2000) Evolution of the 14-3-3 protein family: Does the large number of isoforms in multicellular organisms reflect functional specificity? J. Mol. Evol. 51, 446–458.PubMedGoogle Scholar
  10. 10.
    Fuglsang, A.T., Visconti, S., Drumm, K., et al., (1999) Binding of 14-3-3 protein to the plasma membrane H+-ATPase AHA2 involves the three C-terminal residues Tyr(946)-Thr-Val and requires phosphorylation of Thr(947). J. Biol. Chem. 274, 36,774–36,780.CrossRefGoogle Scholar
  11. 11.
    Svennelid, F., Olsson, A., Piotrowski, M., et al., (1999) Phosphorylation of Thr-948 at the C terminus of the plasma membrane H+-ATPase creates a binding site for the regulatory 14-3-3 protein. Plant Cell. 11, 2379–2391.PubMedCrossRefGoogle Scholar
  12. 12.
    Fuglsang, A. T., Borch, J., Bych, K., Jahn, T. P., Roepstorff, P., Palmgren, M. G. (2003) The binding site for regulatory 14-3-3 protein in plant plasma membrane H+-ATPase- Involvement of a region promoting phosphorylation-independent interaction in addition to the phosphorylation-dependent C-terminal end. J. Biol. Chem. 278, 42,266–42,272.CrossRefGoogle Scholar
  13. 13.
    Baunsgaard, L., Fuglsang, A. T., Jahn, T., Korthout, H. A., de Boer, A. H., Palmgren, M. G. (1998) The 14-3-3 proteins associate with the plant plasma membrane H+-ATPase to generate a fusicoccin binding complex and a fusicoccin responsive system. Plant J. 13, 661–671.PubMedCrossRefGoogle Scholar
  14. 14.
    Moorhead, G., Douglas, P., Cotelle, V., et al. (1999) Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins. Plant J. 18, 1–12.PubMedCrossRefGoogle Scholar
  15. 15.
    Shevchenko, A., M. Wilm, O. Vorm, and M. Mann (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858.PubMedCrossRefGoogle Scholar
  16. 16.
    Kussmann, M., Lassing, U., Sturmer, C. A., Przybylski, M., and Roepstorff, P. (1997) Matrix-assisted laser desorption/ionization mass spectrometry sample preparation techniques designed for various peptide and protein analytes. J. Mass. Spectrom. 32, 593–601.CrossRefGoogle Scholar
  17. 17.
    Jebanathirajah, J.A., Andersen, S., Blagoev, B., and Roepstorff, P. (2002) A rapid screening method to monitor expression of recombinant proteins from various prokaryotic and eukaryotic expression systems using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Anal. Biochem. 305, 242–250.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2006

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdense MDenmark

Personalised recommendations