van Bentum Mirjam, Klinger Bertram, Sieber Anja, Naghiloo Sheyda, Zauber Henrik, Lehmann Nadine, Haji Mohamed, Niquet Sylvia, Mertins Philipp, Blüthgen Nils, Selbach Matthias
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, 13092, Berlin, Germany.
Institute for Theoretical Biology, Faculty of Life Sciences, Humboldt-Universität zu Berlin, Unter den Linden 6, 10099, Berlin, Germany.
Nat Commun. 2025 May 27;16(1):4884. doi: 10.1038/s41467-025-59404-y.
Targeted kinase inhibitors are a cornerstone of cancer therapy, but their success is often hindered by the complexity of cellular signaling networks that can lead to resistance. Overcoming this challenge necessitates a deep understanding of cellular signaling responses. While standard global phosphoproteomics offers extensive insights, lengthy processing times, the complexity of data interpretation, and frequent omission of crucial phosphorylation sites limit its utility. Here, we combine data-independent acquisition (DIA) with spike-in of synthetic heavy stable isotope-labeled phosphopeptides to facilitate the targeted detection of particularly informative phosphorylation sites. Our spike-in enhanced detection in DIA (SPIED-DIA) approach integrates the improved sensitivity of spike-in-based targeted detection with the discovery potential of global phosphoproteomics into a simple workflow. We employed this method to investigate synergistic signaling responses in colorectal cancer cell lines following MEK inhibition. Our findings highlight that combining MEK inhibition with growth factor stimulation synergistically activates JNK signaling in HCT116 cells. This synergy emphasizes the therapeutic potential of concurrently targeting MEK and JNK pathways, as evidenced by the significantly impaired growth of HCT116 cells when treated with both inhibitors. Our results demonstrate that SPIED-DIA effectively identifies synergistic signaling responses in colorectal cancer cells, presenting a valuable tool for uncovering new therapeutic targets and strategies in cancer treatment.
靶向激酶抑制剂是癌症治疗的基石，但其成功往往受到细胞信号网络复杂性的阻碍，而这种复杂性可能导致耐药性。克服这一挑战需要深入了解细胞信号反应。虽然标准的全局磷酸化蛋白质组学提供了广泛的见解，但处理时间长、数据解释复杂以及关键磷酸化位点经常被遗漏限制了其效用。在这里，我们将数据非依赖采集（DIA）与合成重稳定同位素标记的磷酸肽的掺入相结合，以促进对特别有信息价值的磷酸化位点的靶向检测。我们的DIA中掺入增强检测（SPIED-DIA）方法将基于掺入的靶向检测的提高的灵敏度与全局磷酸化蛋白质组学的发现潜力整合到一个简单的工作流程中。我们采用这种方法研究了MEK抑制后结直肠癌细胞系中的协同信号反应。我们的研究结果突出表明，在HCT116细胞中，将MEK抑制与生长因子刺激相结合可协同激活JNK信号。这种协同作用强调了同时靶向MEK和JNK途径的治疗潜力，当用两种抑制剂处理时，HCT116细胞的生长显著受损就证明了这一点。我们的结果表明，SPIED-DIA有效地识别了结直肠癌细胞中的协同信号反应，为揭示癌症治疗中的新治疗靶点和策略提供了一种有价值的工具。
