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. Author manuscript; available in PMC: 2009 Nov 21.
Published in final edited form as: Oncogene. 2009 Apr 13;28(20):2112–2118. doi: 10.1038/onc.2009.71

Figure 3.

Figure 3

MALDI TOF mass spectra of kinase-phosphorylated 10-57 S15(P). p53 10-57 S15(P) peptide was successively phosphorylated by casein kinase I, casein kinase II (New England Biolabs, MA, USA) and c-jun N-terminal kinase JNK (Millipore, MA, USA). Each enzyme phosphorylates three, two and one serine or threonine residue, respectively. Repeated rounds of kinase phosphorylations produced the final product, which is phosphorylated on all available seven Ser/Thr on p53 TAD. The presence of the respective mono- to hepta-phosphorylated peptides are colour-coded onto the mass spectra, with molecular weights shown top left. Mass peaks are broad due to the presence of multiple ionic salt species. Kinase reactions were performed for 24 hours at 30 °C according to the manufacturer’s instructions, using (per mL) 200 μM peptide, 100 μL of 100 mM Mg-ATP, 10 μL enzyme, 2 μL EDTA-free protease inhibitor (Roche, Switzerland) and 1 μL β-mercaptoethanol. The final product was purified as above and consisted mostly of hepta-phosphorylated product as confirmed by MALDI-TOF, with small fractions of lower phospho-species.