Ji Fei, Hur Moises, Hur Sungwon, Wang Siwen, Sarkar Priyanka, Shao Shiqun, Aispuro Desiree, Cong Xu, Hu Yanhao, Li Zhonghan, Xue Min
Department of Chemistry, University of California, Riverside, Riverside, California 92521, United States.
Martin Luther King Jr High School, Riverside, California 92508, United States.
ACS Bio Med Chem Au. 2023 Apr 28;3(3):283-294. doi: 10.1021/acsbiomedchemau.3c00018. eCollection 2023 Jun 21.
Multiplex protein imaging technologies enable deep phenotyping and provide rich spatial information about biological samples. Existing methods have shown great success but also harbored trade-offs between various pros and cons, underscoring the persisting necessity to expand the imaging toolkits. Here we present PACIFIC: photoactive immunofluorescence with iterative cleavage, a new modality of multiplex protein imaging methods. PACIFIC achieves iterative multiplexing by implementing photocleavable fluorophores for antibody labeling with one-step spin-column purification. PACIFIC requires no specialized instrument, no DNA encoding, or chemical treatments. We demonstrate that PACIFIC can resolve cellular heterogeneity in both formalin-fixed paraffin-embedded (FFPE) samples and fixed cells. To further highlight how PACIFIC assists discovery, we integrate PACIFIC with live-cell tracking and identify phosphor-p70S6K as a critical driver that governs U87 cell mobility. Considering the cost, flexibility, and compatibility, we foresee that PACIFIC can confer deep phenotyping capabilities to anyone with access to traditional immunofluorescence platforms.
多重蛋白质成像技术能够实现深度表型分析，并提供有关生物样本的丰富空间信息。现有方法已取得巨大成功，但在各种优缺点之间也存在权衡，这突出表明持续需要扩展成像工具集。在此，我们展示了PACIFIC：具有迭代切割功能的光活性免疫荧光，这是一种新型的多重蛋白质成像方法。PACIFIC通过使用可光切割荧光团进行抗体标记并通过一步式旋转柱纯化实现迭代多重分析。PACIFIC不需要专门的仪器、DNA编码或化学处理。我们证明，PACIFIC可以解析福尔马林固定石蜡包埋（FFPE）样本和固定细胞中的细胞异质性。为了进一步突出PACIFIC如何辅助发现，我们将PACIFIC与活细胞追踪相结合，并确定磷酸化的p70S6K是控制U87细胞迁移的关键驱动因素。考虑到成本、灵活性和兼容性，我们预计PACIFIC可以为任何能够使用传统免疫荧光平台的人赋予深度表型分析能力。
