2005 Volume 49 Issue 3 Pages 73-81
Protein phosphorylation is one of the most significant post-translational modifications that regulate protein functions. In most researches on protein phosphorylation, autoradiography of 32P-labeled proteins and/or western blotting with specific antibodies has been generally employed. In this paper, we assessed the effectiveness of affinity purification of phosphorylated proteins and phosphorylated peptides prior to mass spectrometric analysis. We primarily confirmed that MALDI-TOF-MS is actually effective in assignment of phosphorylation sites on phosphoseryl/phosphothreonyl authentic peptides and tryptic peptides derived from natural casein, whereas this technique is not efficient for phosphotyrosyl peptides. We subsequently optimized the procedure of immobilized metal ion affinity chromatography (IMAC) for enhancement of MALDI-TOF-MS signals in PSD of phosphorylated peptides. We further assessed the effectiveness of affinity concentration of phosphorylated proteins performed prior to electrophoretic separation using a commercially available kit. The optimization of these methods allowed us to assign a novel phosphorylated site on myelin basic protein (MBP) isolated from rat spinal cord.
