Fig. 2. PhosPIB approach identifies differential phosphorylation sites on PPP regulatory proteins.

(A) Schematic of process to identify differential phosphorylation sites on PPP regulatory proteins: PIB eluates from asynchronous or mitotic HeLa cells were digested into peptides, TMT-labeled, and enriched for phosphorylated peptides using Fe-NTA columns prior to LC-MS/MS analysis. (B) Volcano plot of phosphorylation sites identified by phosphopeptide enrichment of PIB eluates from asynchronous or mitotic cells. Sites highlighted in blue significantly decreased and those in red significantly increased in mitotic HeLa PIB eluates (P<0.05, n=3 independent biological replicates). (C) Phosphorylation sites identified on PP1, PP2A, PP4 or PP6 catalytic or regulatory subunits that increased or decreased significantly (red or blue, respectively; P<0.05) in mitotic PIB eluates. (D) Enriched phosphorylation site motifs from phosphopeptides that significantly increased (P < 0.05, log₂ fold change of 1.5) in mitotic or asynchronous PIB eluates. (E) Significantly increased or decreased (P < 0.05) phosphorylation sites from mitotic PIB eluates were classified as either proline-directed, basic (basophilic) or acidic (acidophilic) per surrounding amino acids.