Comparative assessment of quantification methods for tumor tissue phosphoproteomics

The choice of quantification method is critical for study design and comparison of cancer phosphoproteomes. We comparatively assessed label-free quantification (LFQ), spike-in-SILAC (stable isotope labeling of amino acids in cell culture) and tandem mass tag (TMT) labeling techniques for quantitative phosphoproteome analysis in tumor tissues. Using ovarian cancer tissue as an example, our study establishes a resource for the design and analysis of quantitative phosphoproteomics studies in cancer research and diagnosis. TMT provided the lowest accuracy and the highest precision and robustness across different phosphosite abundances and matrices (cell culture versus tumor tissue). Spike-in-SILAC offered the best compromise between these features, but was limited by low phosphosite coverage. LFQ provided the lowest precision, but the highest number of phosphosite identifications. Spike-in-SILAC and LFQ were susceptible to matrix effects arising from different sample types. Analysis based on match between run (MBR) improved phosphosite coverage across technical replicates in LFQ and spike-in-SILAC, but further reduced the precision and robustness of quantification.