An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells.
An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells.
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DOI：
10.1038/s41467-017-01854-0
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发表时间：
2017-11-24
影响因子：
16.6
通讯作者：
Moellering RE
中科院分区：
文献类型：
--
作者：
Li G;Montgomery JE;Eckert MA;Chang JW;Tienda SM;Lengyel E;Moellering RE
将化学探针整合到蛋白质组学工作流程中可以询问蛋白质活性，而不是丰度。由于样品均质化、信号平均化和对丰富蛋白质的偏好，目前的方法限制了可以解决的生物学背景。在这里，我们报告了一个平台，该平台将全家族的化学探针与邻近依赖性寡核苷酸扩增和成像相结合，以高空间分辨率在天然环境中定量酶活性。这种方法，活性依赖性邻近连接（ADPL），丝氨酸水解酶和半胱氨酸蛋白酶的应用，使定量的差异酶活性的内源性变化，在本地化和表达。在竞争性形式中，可以定量小分子靶标与活细胞中的内源性蛋白质的接合。最后，样品结构的保留使得能够询问复杂的环境，例如细胞共培养和患者样品。ADPL应该适合于不同的探针和蛋白质家族，以当前方法无法达到的规模和分辨率检测活性酶。酶活性的询问涉及许多细胞的整体平均，空间关系的损失，并且通常偏向于丰富的蛋白质。在这里，作者开发了活性依赖性邻近连接，以在相关生物学背景下在细胞和亚细胞水平上量化酶活性。
Integration of chemical probes into proteomic workflows enables the interrogation of protein activity, rather than abundance. Current methods limit the biological contexts that can be addressed due to sample homogenization, signal-averaging, and bias toward abundant proteins. Here we report a platform that integrates family-wide chemical probes with proximity-dependent oligonucleotide amplification and imaging to quantify enzyme activity in native contexts with high spatial resolution. Application of this method, activity-dependent proximity ligation (ADPL), to serine hydrolase and cysteine protease enzymes enables quantification of differential enzyme activity resulting from endogenous changes in localization and expression. In a competitive format, small-molecule target engagement with endogenous proteins in live cells can be quantified. Finally, retention of sample architecture enables interrogation of complex environments such as cellular co-culture and patient samples. ADPL should be amenable to diverse probe and protein families to detect active enzymes at scale and resolution out of reach with current methods. The interrogation of enzyme activity involves the ensemble averaging of many cells, loss of spatial relationships and is often biased to abundant proteins. Here the authors develop activity-dependent proximity ligation to quantify enzyme activity at the cellular and sub-cellular level in relevant biological contexts.
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影响因子：
9.2
作者：
Bright, Nicholas A.;Davis, Luther J.;Luzio, J. Paul
通讯作者：
Luzio, J. Paul
影响因子：
46.9
作者：
Fredriksson, S;Gullberg, M;Landegren, U
通讯作者：
Landegren, U
影响因子：
14.8
作者：
Lemke, Edward A.;Schultz, Carsten
通讯作者：
Schultz, Carsten
影响因子：
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通讯作者：
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影响因子：
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作者：
Chang JW;Nomura DK;Cravatt BF
通讯作者：
Cravatt BF