TABLE 1.
List of C-tail MOP receptor phosphorylated and unphosphorylated peptides identified by nanoLC-MS/MS CID and/or ETD analysis from cells treated for 20 min at room temperature with KRH buffer alone (CTRL), 1 μm DAMGO, or 1 μm 1DMe
The abbreviations used are as follows: Theo. Mass, theoretical mass; MC, missed cleavage.
| Theo. Mass | MC | Position | Sequence | Phospho-residue | Present in sample |
Agonist effect | DAMGO/1DMe | ||
|---|---|---|---|---|---|---|---|---|---|
| CTRL | DAMGO | 1DMe | |||||||
| 2009.9113 | 0 | 351–367 | EFCIPTSSNIEQQNSTR | √ | √ | √ | |||
| 2089.8776 | 0 | 351–367 | EFCIPTSSNIEQQNSTR | Ser-365 | √ | √a | √a | No increase | 1.1 |
| 2089.8776 | 0 | 351–367 | EFCIPTSSNIEQQNSTR | Ser-358 | √ | ||||
| 1211.5531 | 0 | 374–384 | DHPSTANTVDR | √ | √ | √ | |||
| 1291.5194 | 0 | 374–384 | DHPSTANTVDR | Ser-377 or Thr-378 | √ | √ | √ | Increase | 1.9 |
| 1371.4857 | 0 | 374–384 | DHPSTANTVDR | Ser-377 + Thr-378 | √ | √ | Increase | 6.6 | |
| 1710.8034 | 1 | 370–384 | QNTRDHPSTANTVDR | √ | √ | √ | |||
| 1790.7697 | 1 | 370–384 | QNTRDHPSTANTVDR | Thr-372 | √ | √ | √ | Decrease | 0.4 |
| 1870.7360 | 1 | 370–384 | QNTRDHPSTANTVDR | Thr-372 + Ser-377 or Thr-378 | √ | √ | √ | Increase | 2.6 |
| 1950.7023 | 1 | 370–384 | QNTRDHPSTANTVDR | Thr-372 + Ser-377 + Thr-378 | √ | √ | Increase | 20.1 | |
| 2644.3530 | 0 | 385–408 | TNHQLENLEAETVPLPLQGPMVSK | √ | √ | √ | |||
a No distinction between Ser-365 and Thr-366 was obtained for the phosphorylation site. All MS/MS spectra of phosphorylated peptides were manually interpreted and are displayed in supplemental Fig. 4. For each peptide, the normalized DAMGO/1DMe relative abundance ratios were calculated from peak area automatically extracted using the label-free module of the in-house developed MFPaQ version 4.0.0 software as described under “Experimental Procedures.”