Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated in cancer. As a result, profiling tumor pTyr levels may reveal therapeutic insights critical to combating disease. Existing discovery and targeted mass spectrometry-based methods used to monitor pTyr networks involve a tradeoff between broad coverage of the pTyr network, reproducibility in target identification across analyses, and accurate quantification. To address these limitations, we developed a targeted approach, termed SureQuant pTyr, coupling low input pTyr enrichment with a panel of isotopically labeled, tyrosine phosphorylated internal standard (IS) peptides. Using internal standard guided acquisition, the real-time detection of IS peptides during the analysis initiates the sensitive and selective quantitation of endogenous pTyr targets. This framework allows for reliable quantification of several hundred commonly dysregulated pTyr targets with high quantitative accuracy, enhances target detection success rates, and improves the robustness and usability of targeted acquisition. We establish the clinical applicability of SureQuant pTyr by profiling pTyr signaling levels in human colorectal tumors using minimal sample input, characterizing patient specific oncogenic driving mechanisms. While in some cases pTyr profiles align with previously reported proteomic, genomic, and transcriptomic molecular characterizations, we highlight instances of new insights gained using pTyr characterization and emphasize the complementary nature of pTyr measurements with traditional biomarkers for improving patient stratification and identifying therapeutic targets. The turn-key nature of this approach opens the door to rapid and reproducible pTyr profiling in research and clinical settings alike and enable pTyr-based measurements for applications in precision medicine.

中文翻译：

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高密度，酪氨酸磷酸化的靶向监测揭示了人类肿瘤中激活的信号网络

酪氨酸磷酸化（pTyr）在信号转导中起关键作用，通常在癌症中失调。结果，对肿瘤pTyr水平进行分析可能会揭示对对抗疾病至关重要的治疗见解。用于监视pTyr网络的现有发现和基于目标质谱的方法涉及在pTyr网络的广泛覆盖范围，整个分析中目标识别的可重复性与精确定量之间的权衡。为了解决这些限制，我们开发了一种靶向方法，称为SureQuant pTyr，将低输入pTyr富集与一组同位素标记的酪氨酸磷酸化内标（IS）肽偶联。使用内部标准指导的采集，分析过程中IS肽的实时检测启动了对内源性pTyr靶标的灵敏和选择性定量。该框架可实现对数百种通常失调的pTyr靶标的可靠定量，并具有很高的定量准确度，提高了靶标检测成功率，并提高了目标采集的鲁棒性和可用性。我们通过使用最少的样本输入来分析人结肠直肠肿瘤中的pTyr信号水平，从而表征患者特定的致癌驱动机制，从而建立SureQuant pTyr的临床适用性。在某些情况下，pTyr谱与先前报道的蛋白质组学，基因组和转录组学分子特征相符，我们重点介绍了使用pTyr表征获得的新见解的实例，并强调了pTyr测量与传统生物标记物的互补性质，以改善患者分层和确定治疗靶标。这种方法的交钥匙性质为在研究和临床环境中快速，可重复的pTyr分析打开了大门，并使基于pTyr的测量可用于精密医学。