Abstract
Protein phosphorylation is a critical mechanism in the regulation of cellular biochemical pathways and phosphopeptides can play an important role in determining function. However, the use of phosphopeptides especially multiphosphorylated peptides is hampered by their low abundance, difficulty in isolation from biological samples and in their chemical synthesis. Here we describe methodologies for the Fmoc synthesis, purification and mass spectral analysis of the multiphosphorylated sequence H-[Asp-(Ser(P))2]3-Asp-OH from phosphophoryn a protein involved in dentine mineralization. Critical steps in the synthesis of phosphophoryn using Fmoc-Ser(PO3Bzl,H)-OH as the building block were double acylation steps for each residue, alternating HBTU and HATU as the acylating agents and synthesis on a chlorotrityl resin which was essential for complete removal of the benzyl-side chain protecting groups. The synthetic phosphophoryn was only effectively purified by anion exchange and size exclusion chromatography as both alkaline and acid buffers failed to aid in purification by reversed phase HPLC. MALDI-TOF analysis of phosphophoryn was achieved with good sensitivity (20 fmol/ml) and resolution using the DNA matrix 3-hydroxypicolinic acid, whereas typical protein/peptide matrices failed to provide mass spectra. The synthetic phosphophoryn peptide was found to bind calcium, binding 6 mol of calcium per mole of peptide. In conclusion the methodology described here can be easily adopted for the synthesis and analysis of a wide variety of multiphosphorylated peptides.
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Acknowledgements
The Australian National Health and Medical Research Council (Project No. 251707) supported this work. Neil M. O’Brien-Simpson is a CR Roper fellow.
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O’Brien-Simpson, N.M., Attard, T.J., Loganathan, A. et al. Synthesis and Characterisation of a Multiphosphorylated Phosphophoryn Repeat Motif; H-[Asp-(Ser(P))2]3-Asp-OH. Int J Pept Res Ther 13, 469–478 (2007). https://doi.org/10.1007/s10989-007-9106-z
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DOI: https://doi.org/10.1007/s10989-007-9106-z