Identification and semi-quantitation of phosphorylation sites on Schizosaccharomyces pombe DNA-binding protein Rap1. A Rap1 construct was cultured in homozygous diploid temperature-sensitive pat1-114 mutant strain of S. pombe cells (time points: 3.5 h and 4.5h), and was purified by anti-PK immunoprecipitation. The resulting samples were run into a 1D SDS-PAGE gel with the target band excised, tryptically digested and analysed by LC-MS/MS using a Velos Orbitrap mass spectrometer.