Table 1 Phosphorylation site analysis of Bad. Phosphorylated peptides were eluted from the HPLC (Fig 2) were identified by ms/ms mass spectrometry. The amino acid numbers of the peptide in murine Bad are indicated and the phosphorylation site underlined. Results for the release of 32P during Edman degradation cycle sequencing and phospho amino acid analysis of these fractions are also indicated. The percentage of 32P obtained in each HPLC peak, relative to the total 32P eluted in all the peaks is also listed (last column)
Kinase | Peak | Residue Numbers | Peptide Sequence | Phospho-amino acid analysis | Solid Phase cycle no. | Phosphorylation site | % 32P |
|---|---|---|---|---|---|---|---|
Pim-1 | 1 | 154 – 163 | MS DEFEGSFK | Phospho Ser | 2 | Ser 155 | 11 |
|  | 2 | 136 – 146 | S APPNLWAAQR | Phospho Ser | 1 | Ser 136 | 15 |
|  | 3 | 110 – 131 | HSS YPAGTEEDEGMEEELSPFR | Phospho Ser | 3 | Ser 112 | 60 |
|  | 4 | 110 – 131 | HSS YPAGTEEDEGMEEELSPFR | Phospho Ser | 3 | Ser 112 | 14 |
Pim-2 | 3 | 110 – 131 | HSS YPAGTEEDEGMEEELSPFR | Phospho Ser | 3 | Ser 112 | 82 |
|  | 4 | 110 – 131 | HSS YPAGTEEDEGMEEELSPFR | Phospho Ser | 3 | Ser 112 | 18 |
Pim-3 | 1 | 154 – 163 | MS DEFEGSFK | Phospho Ser | 2 | Ser 155 | 14 |
|  | 2 | 136 – 146 | S APPNLWAAQR | Phospho Ser | 1 | Ser 136 | 28 |
|  | 3 | 110 – 131 | HSS YPAGTEEDEGMEEELSPFR | Phospho Ser | 3 | Ser 112 | 27 |
|  | 4 | 110 – 131 | HSS YPAGTEEDEGMEEELSPFR | Phospho Ser | 3 | Ser 112 | 6 |
| Â | 5 | unidentified | unidentified | unidentified | N/A | unidentified | 13 |
|  | 6 | 170 – 178 | S AGTATQMR | Phospho Ser | 1 | Ser 170 | 4 |
|  | 7 | 92 – 107 | AAT NS HHGGAGAMETR | Phospho Thr/Ser | 3, 5 | Thr 94 Ser 96 | 2 |
|  | 8 | 170 – 178 | S AGT ATQMR | Phospho Ser/Thr | 1, 4 | Ser 170 Thr 173 | 2 |
| Â | 9 | unidentified | unidentified | unidentified | N/A | unidentified | 3 |