Samples of CLH-3b protein isolated from cells expressing CLH-3b in the presence of either active GCK-3 or KD GCK-3 were evaluated by targeted MS/MS analysis (see Materials and methods). Peak areas were determined for each of the modified SITHLSFGR peptides indicated and the peak areas for the unmodified reference peptides KILTVEEK, LVHGSSGGIFENESR, and YVDSQIGTK. The peak areas for the phosphorylated SITHLSFGR peptide were then normalized to the peak areas for each of the unmodified reference peptides. Relative phosphorylation was calculated as the ratio of peak areas of the SITHLSFGR peptide from cells expressing functional GCK-3 or KD GCK-3. Values are means ± SD for three separate MS runs.

Phosphorylation can be identified by an increase in peptide mass of +80, which corresponds to the addition of a phosphate group (HPO₃). It can also be identified by the neutral loss of H₃PO₄ from the phosphorylated peptide. When compared to a peptide that is not phosphorylated, a net decrease in peptide mass of −18 is detected. (−18) and (80) refer to the peptide mass changes that were observed. The MS/MS/MS spectra of the neutral loss of phosphoric acid from the singly phosphorylated peptide allowed us to distinguish the phosphorylation at S747 versus S742 (see Figs. S1, B–D).