Technology-driven the advance of phosphoproteomics- A highly sensitive kinase motif-targeting phosphoproteomic strategy for phosphorylation signaling network elucidation

Abstract

The pathway specificity of the phosphorylation-mediated signaling cascade is orchestrated by interaction between protein kinases and a variety of its downstream molecules. Despite the analytical approaches for phosphoproteomics has matured within the last decade, enrichment and detection towards comprehensive phosphoproteomic profiling with limited sample amount remain further improvement. Besides, it is still difficult for conventional phosphoproteomic approaches to distinguish whether the fold-change of phosphopeptides is induced at the phosphorylation level or is caused by the change at the expression level of proteins. Furthermore, two-fold changes in absolute values of phosphorylation stoichiometry such as 0.5% to 1% or 50% to 100% may represent fundamentally different cellular functions. Here, we firstly developed a StageTip based phosphoproteomic assay which minimizing the steps of sample transfer and increasing the detection sensitivity. By using meter-long reversed phase monolithic silica column, over 10,000 phosphopeptides can be detected from only 150 ug tryptic peptides. We have further developed a highly sensitive kinase motif targeting proteomic assay for specific kinase substrate purification. Up to 6490 motif targeting tyrosine sites and 4026 S/T sites can be detected via kinase reaction including 1 acidic, 2 basophilic, 3 Pro-directed and 5 tyrosine kinases. Through sequence motif analysis for identified motif targeting phosphopeptides, the specificity of kinase recognition was higher in S/T kinase than tyrosine kinase. Finally, we can measure the phosphorylation stoichiometry of 7392 phosphorylation sites including 2795 low-abundance tyrosine phosphorylation sites by integrating dephosphorylation and isotope tagging with kinase motif targeting proteomic assay. Comparing gefitinib-resistant and sensitive lung cancer cells, we reveal that post-translational phosphorylation changes are significantly more dramatic than those at the protein and messenger RNA levels, and find potential drug targets within the kinase–substrate network associated with acquired drug resistance.