Fig. 7

Identification of phosphorylation sites of mMDH1 treated with CDKA;1-CYCD2;1 complexes in vitro. a Gel image of HisGST-mMDH1 subjected to mass spectrometry analyses. The mMDH1 proteins were treated without (−) and with (+) CDKA;1-CYCD2;1. The proteins were separated by SDS-PAGE after the kinase reaction and stained with coomassie brilliant blue. b Mass chromatograms (top) of the selected peptide and mass spectrum (bottom) of the peptide. Corresponding to (c), the non-phosphorylated peptide 116-DDLFNINAGIVK-127 was detected in the both samples of mMDH1 treated with (red) and without (blue) CDKA;1-CYCD2;1 complexes. c Mass chromatograms (top) of the selected peptide and mass spectrum (bottom) of the peptide. The phosphopeptide 110-KPGM(ox)T(ph)RDDLFNINAGIVK-127 was detected only in the sample of mMDH1 treated with CDKA;1-CYCD2;1 (red), but not in the sample without kinase (blue). T(ph) and M(ox), in the peptide sequence, indicate the phosphorylated threonine and the oxidized methionine, respectively. The underline in the peptide sequence indicates the peptide sequence found in (b). d MS/MS spectra of a phosphopeptide (110- KPGM(ox)T(ph)RDDLFNINAGIVK-127) from mMDH1 treated with CDKA;1-CYCD2;1. The b and y ion series represent fragment ions containing the N- and C-termini of the peptide, respectively. Mass chromatogram (b and c, top) is given by plotting the x-axis as the retention time and the y-axis as the ion peak intensity. Mass spectrum (b and c, bottom) is given by plotting the x-axis as the mass-to-charge ratio (m/z) and the y-axis as the ion peak intensity