Scalable phosphotyrosine enrichment with SH2 superbinder enables deep profiling of EGF responses.

Phosphotyrosine signaling plays a critical role in many biological processes, from cell proliferation to immune response. Despite its importance, proteomic studies of tyrosine phosphorylation have been limited in scale and throughput due to the need for specialized enrichment with costly reagents and labor-intensive protocols. To address these challenges, we developed R2HaPpY, a phosphotyrosine enrichment method that combines highly simplified phosphotyrosine superbinder reagent preparation and automated high-throughput enrichment. Our new reagent binds phosphotyrosine peptides at higher efficiency than other enrichment reagents and reduces both cost and preparation time by 20-fold. We generalized the R2HaPpY method to samples of low and high levels of phosphotyrosine. We benchmarked biological application to study EGF signaling dynamics in HeLa cells. Using only ~1 mg of input peptides, we detect and quantify 1651 unique phosphotyrosine sites. These include 878 regulated pY sites, many of which are novel or not previously annotated as EGF-responsive. Our results reveal differential temporal regulation and represent the largest phosphotyrosine dataset of cellular response to EGF stimulation to date. This streamlined, cost-effective, and sensitive method enables quantitative mapping of tyrosine phosphorylation dynamics at a scale of hundreds of samples, facilitating integration of phosphotyrosine signaling into multiomic studies across diverse biological systems and disease states.