Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.

中文翻译：

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冷冻切片中丝氨酸水解酶活性的高分辨率共聚焦荧光成像——在胶质瘤脑中的应用揭示了源自肿瘤相关中性粒细胞的活动热点

丝氨酸水解酶 (SHs) 是功能多样的酶家族，在健康和疾病中发挥着关键作用，并已成为许多临床病症中的重要治疗靶点。在揭示 SH 在各种生物系统中的作用的研究中，使用氟膦酸盐 (FP) 探针的基于活性的蛋白质分析 (ABPP) 一直是一种强大的化学蛋白质组学方法。ABPP 利用细胞/组织蛋白质组和 FP 弹头，连接到用于凝胶内荧光成像的荧光报告器或用于链霉亲和素富集和基于 LC-MS/MS 的目标识别的生物素标签。现有的 ABPP 方法基于凝胶中的移动性或基于 MS 的目标识别来表征全局 SH 活性，并且不能揭示负责源自复杂蛋白质组的单个 SH 活性的细胞类型的身份。这里，通过使用对 SH 家族具有广泛反应性的活性探针，我们将 ABPP 方法推进到胶质瘤脑冷冻切片，首次实现了肿瘤微环境中全局 SH 活性的高分辨率共聚焦荧光成像。通过对活性探针标记的切片进行广泛的免疫组织化学来鉴定肿瘤相关细胞类型。组织-ABPP 表明胶质瘤与正常脑中的 SH 活性升高，并揭示了源自肿瘤相关中性粒细胞 (TAN) 而不是肿瘤相关巨噬细胞 (TAM) 的活动热点。基于平行凝胶的 ABPP 与基于 LC-MS/MS 的目标验证相结合，提供了彻底的优化和验证。我们的研究将 ABPP 方法推进到组织切片，在解剖学保存的复杂原生细胞环境中实现全局 SH 活动的高分辨率共聚焦荧光成像。为了在整个部分中实现 SH 活动的全局画像，采用了对 SH 家族成员具有广泛反应性的探针。由于 ABPP 不需要目标身份的先验知识，我们无法想象为什么目前描述的方法不适用于切片，无论物种和组织来源如何。