Mass cytometry permits the high dimensional analysis of complex biological samples; however, some techniques are not yet integrated into the mass cytometry workflow due to reagent availability. The use of self-labeling protein systems, such as HaloTag, are one such application. Here, we describe the design and implementation of the first mass cytometry ligands for use with HaloTag. “Click”-amenable HaloTag warheads were first conjugated onto poly(l-lysine) or poly(acrylic acid) polymers that were then functionalized with diethylenetriaminepentaacetic acid (DTPA) lutetium metal chelates. Kinetic analysis of the HaloTag labeling rates demonstrated that the structure appended to the 1-chlorohexyl warhead was key to success. A construct with a diethylene glycol spacer appended to a benzamide gave similar rates (k_obs ∼ 10² M^–1 s^–1), regardless of the nature of the polymer. Comparison of the polymer with a small molecule chelate having rapid HaloTag labeling kinetics (k_obs ∼ 10⁴ M^–1 s^–1) suggests the polymers significantly reduced the HaloTag labeling rate. HEK293T cells expressing surface-exposed GFP-HaloTag fusions were labeled with the polymeric constructs and ¹⁷⁵Lu content measured by cytometry by time-of-flight (CyTOF). Robust labeling was observed; however, significant nonspecific binding of the constructs to cells was also present. Heavily pegylated polymers demonstrated that nonspecific binding could be reduced to allow cells bearing the HaloTag protein to be distinguished from nonexpressing cells.

中文翻译：

---

质谱流式细胞术可检测 HaloTag 配体的设计参数

质谱流式分析仪可以对复杂的生物样品进行高维分析；然而，由于试剂的可用性，一些技术尚未集成到质谱流式细胞术工作流程中。使用自标记蛋白质系统（例如 HaloTag）就是这样的应用之一。在这里，我们描述了第一个与 HaloTag 一起使用的质量流式细胞术配体的设计和实现。适合“点击”的 HaloTag 弹头首先缀合到聚（l-赖氨酸）或聚（丙烯酸）聚合物上，然后用二亚乙基三胺五乙酸（DTPA）镥金属螯合物进行功能化。HaloTag 标记率的动力学分析表明，附加到 1-氯己基弹头的结构是成功的关键。无论聚合物的性质如何，在苯甲酰胺上附加二甘醇间隔基的构建体都给出相似的速率（k _obs ∼ 10 ² M ^–1 s ^{–1 ）。}将聚合物与具有快速 HaloTag 标记动力学 ( k _obs ∼ 10 ⁴ M ^–1 s ^–1 )的小分子螯合物进行比较表明，聚合物显着降低了 HaloTag 标记率。用聚合物构建体标记表达表面暴露的 GFP-HaloTag 融合体的 HEK293T 细胞，并通过飞行时间 (CyTOF) 流式细胞术测量^{175 Lu 含量。}观察到稳健的标记；然而，也存在构建体与细胞的显着非特异性结合。高度聚乙二醇化的聚合物表明，可以减少非特异性结合，从而将携带 HaloTag 蛋白的细胞与非表达细胞区分开来。