A New Workflow for the Analysis of Phosphosite Occupancy in Paired Samples by Integration of Proteomics and Phosphoproteomics Data Sets.

Knowledge of phosphosite occupancy is important to explain biological functions for phosphoproteomics studies. Determination of occupancy using three ratios, i.e., protein ratio, phosphopeptide ratio, and its unmodified peptide counterpart ratio between a pair of samples, is straightforward but suffers from large variances. Here, an optimized protocol of offline fractionation and LC-MS analysis combined with an integrated data processing approach was developed to improve the reliability of the phosphosite occupancy determination. An outlier score S was introduced to evaluate the deviation between the ratio of absolute occupancy and relative occupancy and was further used to define the bounds of a credible interval of absolute occupancy. For a preset product-moment correlation coefficient, the credible interval can be resolved through the S value. Using this strategy, more than 176k unique peptide sequences covering 11k protein groups and 32k phosphosites were identified from one paired hepatocellular carcinoma (HCC) sample and about 3000 reliable phosphosite occupancies were finally determined. By bioinformatics analysis, we characterized the biological properties associated with phosphorylation sites with different quantified occupancies from the paired HCC sample. Data are available via ProteomeXchange with identifier PXD019045.