Mass Spectrometry-based Methods for Phosphorylation Site Mapping of Hyperphosphorylated Proteins Applied to Net1, a Regulator of Exit from Mitosis in Yeast*

protein phosphatase is sequestered within the nucleolus and inhibited by Net1, a component of the RENT complex in budding yeast. During anaphase the RENT complex disassembles, allowing Cdc14 to migrate to the nucleus and cytoplasm where it catalyzes exit from mitosis. The mechanism of Cdc14 release appears to involve the polo-like kinase Cdc5, which is capable of promoting the dis-sociation of a recombinant Net1Cdc14 complex in vitro by phosphorylation of Net1. We report here the phospho-rylation site mapping of recombinant Net1 (Net1N) and a mutant Net1N allele (Net1N-19m) with 19 serines or threonines mutated to alanine. A variety of chromato-graphic and mass spectrometric-based strategies were used, including immobilized metal-affinity chromatogra-phy, alkaline phosphatase treatment, matrix-assisted la-ser-desorption post-source decay, and a multidimen-sional electrospray mass spectrometry-based approach. No one approach was able to identify all phosphopeptides in the tryptic digests of these proteins. Most notably, the presence of a basic residue near the phosphorylated res-idue significantly hampered the ability of alkaline phos-phatase to hydrolyze the phosphate moiety. A major goal of research in proteomics is to identify all proteins and their interactions and post-translational modification states. The failure of any single method to identify all sites in highly phosphorylated Net1N, however, raises signifi-cant concerns about how feasible it is to map phospho-rylation sites throughout the proteome using existin