Chang Alexis, Rodriguez-Mias Ricard A, Berg Matthew D, Moggridge Sophie, Villén Judit
Department of Genome Sciences, University of Washington, Seattle, USA, 98105.
bioRxiv. 2025 May 17:2025.05.14.653984. doi: 10.1101/2025.05.14.653984.
Phosphotyrosine signaling plays a critical role in many biological processes, from cell proliferation to immune response. Despite its importance, systems-level analysis of phosphotyrosine signaling remains a challenge due to costly enrichment reagents and labor-intensive protocols. We previously established an automated phosphotyrosine enrichment method for preparing 96 samples in parallel. Here, we further optimize this method by fusing an SH2 phosphotyrosine superbinder to the HaloTag protein. This allows simple and cost-effective preparation of enrichment beads directly from bacterial lysate, expediting reagent preparation from days to hours. Additionally, our new reagent binds phosphotyrosine peptides at higher efficiency than other enrichment reagents. Using this reagent, we detect and quantify 1,651 unique phosphotyrosine sites from EGF stimulated HeLa cells using only ~1 mg of input peptides per replicate. These include 878 regulated pY sites, many of which are low abundance and not previously detected or annotated as EGF-responsive. This streamlined and sensitive method facilitates comprehensive, quantitative mapping of tyrosine phosphorylation dynamics, enabling broader integration of phosphotyrosine signaling into multiomic and network-level models across diverse biological systems and disease states.
磷酸酪氨酸信号传导在从细胞增殖到免疫反应的许多生物过程中起着关键作用。尽管其很重要，但由于昂贵的富集试剂和劳动密集型方案，对磷酸酪氨酸信号传导进行系统水平的分析仍然是一项挑战。我们之前建立了一种用于并行制备96个样品的自动化磷酸酪氨酸富集方法。在此，我们通过将SH2磷酸酪氨酸超级结合蛋白与HaloTag蛋白融合来进一步优化该方法。这使得能够直接从细菌裂解物中简单且经济高效地制备富集珠，将试剂制备时间从数天缩短至数小时。此外，我们的新试剂比其他富集试剂更高效地结合磷酸酪氨酸肽段。使用该试剂，我们仅通过每个重复样本约1毫克的输入肽段，就检测并定量了来自表皮生长因子（EGF）刺激的HeLa细胞的1651个独特的磷酸酪氨酸位点。其中包括878个受调控的磷酸酪氨酸位点，其中许多是低丰度的，以前未被检测到或未被注释为对EGF有反应。这种简化且灵敏的方法有助于全面、定量地绘制酪氨酸磷酸化动态图谱，使磷酸酪氨酸信号传导能够更广泛地整合到跨多种生物系统和疾病状态的多组学和网络水平模型中。
