Combinations of SPR and MS for characterization of native and recombinant proteins in cell lysates
- 165 Downloads
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 EntriesSurface plasmon resonance/mass spectrometry (SPR/MS) 14-3-3 proteins affinity purification recombinant
Unable to display preview. Download preview PDF.
- 2.Fägerstam, L.G. (1991) A non-label technology for real-time biospecific interaction analysis. Tech. Prot. Chem. II, 65–71.Google Scholar
- 4.Buijs, J. and Franklin, G.C. (2005) SPR-MS in functional proteomics. Brief Funct. Genomic Proteomic.Google Scholar
- 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
- 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