Fig. 5.

Quantitative analysis of the phosphorylation state of Ser-1126 phosphorylation from human separase protein as a function of cell cycle. (A) Percent of phosphorylated Ser-1126 measured before, during, and after exit from mitosis in HeLa cells (▪). Percent of cells in G₂/M phase of the cell cycle was also determined by fluorescence-activated cell sorting analysis (⋄). Separase is quantitatively (>98%) phosphorylated at the anaphase-metaphase transition (nocodozole arrest). The percent of phosphorylated Ser-1126 decreases after mitosis. (B) LC–SRM analysis of phosphorylated and nonphosphorylated separase Ser-1126. These data were acquired from the equivalent of only 16 μg of starting material and 10 fmol of each AQUA peptide on a high-resolution triple quadrupole mass spectrometer. The calculated percent of phosphorylation was 34%. Some of the data in this figure have been published previously. [Reproduced with permission from ref. 15 (Copyright 2001, Elsevier Science).]