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首页> 美国卫生研究院文献>other >Activity-Based Probes linked with Laser-Cleavable Mass Tags for Signal Amplification in Imaging Mass Spectrometry: Analysis of Serine Hydrolase Enzymes in Mammalian Tissue
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Activity-Based Probes linked with Laser-Cleavable Mass Tags for Signal Amplification in Imaging Mass Spectrometry: Analysis of Serine Hydrolase Enzymes in Mammalian Tissue

机译：基于活性的探针与激光切割质量标签连接用于成像质谱中的信号放大：哺乳动物组织中丝氨酸水解酶的分析

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摘要

A novel functional Imaging Mass Spectrometry technology is described that utilizes activity-based probes for imaging enzyme active sites in tissue sections. We demonstrate this technology using an activity-based probe (fluorophosphate) that is specific for serine hydrolases. A dendrimer containing multiple mass tags that is attached to the activity-based probe is used to analyze the binding sites of the probe through release and measurement of the mass tags on laser irradiation. A generation 8 Poly(amido amine) dendrimer with 1024 amino groups was labeled with an azide group and then more than 900 mass tags were attached in order to achieve signal amplification of nearly three orders of magnitude. The experimental protocol first involves binding of the activity-based probe containing an alkyne group to serine hydrolases in the tissue section followed by attachment of the dendrimer labeled with mass tags to the bound probe by Click chemistry. On irradiation of the labeled tissue by the laser beam in a raster pattern, the mass tags are liberated and recorded by the mass analyzer, consequently, the ion image of the mass tag reveals the distribution of serine hydrolases in the tissue. This process was shown using rat brain and mouse embryo sections. Targeted imaging has the advantage of providing high spatial resolution and high sensitivity through the use of signal amplification chemistry with high target specificity through the use of an enzyme activity probe.
机译：描述了一种新型功能成像质谱技术，其利用基于活性的探针在组织切片中成像酶活性部位。我们使用特异于丝氨酸水解酶的活性探针（荧光磷酸）来证明该技术。含有附着于基于活性探针的多个质量标签的树枝状聚合物用于通过释放和测量激光照射的质量标签来分析探针的结合位点。具有1024个氨基的8个聚（amido胺）树枝状聚合物用叠氮基团标记，然后附着大于900个质量标签以实现近三个数量级的信号放大。实验方案首先涉及将含有炔基的基于活性的探针结合到组织截面中的丝氨酸水解酶，然后通过点击化学将标记的树枝状聚合物连接到结合的探针中。在光栅图案中由激光束照射标记的组织，由质量分析仪释放并记录质量标签，因此质量标签的离子图像揭示了组织中丝氨酸水解酶的分布。使用大鼠脑和小鼠胚胎切片显示该过程。目标成像通过使用通过使用酶活性探针，通过使用具有高靶特异性的信号放大化学来提供高空间分辨率和高灵敏度的优点。

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