Ultrasensitive, multiplexed chemoproteomic profiling with soluble activity-dependent proximity ligation
Ultrasensitive, multiplexed chemoproteomic profiling with soluble activity-dependent proximity ligation
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DOI：
10.1073/pnas.1912934116
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发表时间：
2019-10-22
影响因子：
11.1
通讯作者：
Moellering, Raymond E.
中科院分区：
文献类型：
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作者：
Li, Gang;Eckert, Mark A.;Moellering, Raymond E.
化学蛋白质组学方法可以直接报告内源性的活性酶群，这与单独测量转录物或蛋白质丰度有很大不同。全家族探针接触的检测和定量通常需要LC-MS/MS或基于凝胶的检测方法，这些方法存在分辨率低、输入蛋白质组要求高、样品制备费力和设备昂贵等问题。因此，能够利用全家族化学探针的广泛目标分析能力，同时能够对天然样品中的蛋白质活性进行特异性、快速和超灵敏定量的方法，将对基础、转化和临床蛋白质组学应用有用。在这里，我们开发并应用了一种称为可溶性活性依赖邻近连接（sADPL）的方法，该方法利用全家族化学探针将活性酶水平转化为可扩增的条形码寡核苷酸信号。我们证明，与定量PCR信号检测相结合的sADPL能够直接在整个未分离蛋白质组的图形水平上对多个蛋白质家族的活性酶水平进行多重“写入”和“读取”。竞争格式的sADPL分析允许高度敏感的药物-蛋白质相互作用分析检测，这允许体外和体内靶向和脱靶药物参与的直接定量测量。最后，我们证明了比较sADPL分析可以应用于原发人类肿瘤样本的高通量分子表型，从而发现特定肿瘤区室中代谢和蛋白水解酶活性与患者预后之间的新联系。我们期望这种模块化和多路复用的化学蛋白质组学平台将成为药物靶点参与的一般方法，以及用于基础和临床应用的比较酶活性分析。
Chemoproteomic methods can report directly on endogenous, active enzyme populations, which can differ greatly from measures of transcripts or protein abundance alone. Detection and quantification of family-wide probe engagement generally requires LC-MS/MS or gel-based detection methods, which suffer from low resolution, significant input proteome requirements, laborious sample preparation, and expensive equipment. Therefore, methods that can capitalize on the broad target profiling capacity of family-wide chemical probes but that enable specific, rapid, and ultrasensitive quantitation of protein activity in native samples would be useful for basic, translational, and clinical proteomic applications. Here we develop and apply a method that we call soluble activity-dependent proximity ligation (sADPL), which harnesses family-wide chemical probes to convert active enzyme levels into amplifiable barcoded oligonucleotide signals. We demonstrate that sADPL coupled to quantitative PCR signal detection enables multiplexed "writing" and "reading" of active enzyme levels across multiple protein families directly at picogram levels of whole, unfractionated proteome. sADPL profiling in a competitive format allows for highly sensitive detection of drug-protein interaction profiling, which allows for direct quantitative measurements of in vitro and in vivo on- and off-target drug engagement. Finally, we demonstrate that comparative sADPL profiling can be applied for high-throughput molecular phenotyping of primary human tumor samples, leading to the discovery of new connections between metabolic and proteolytic enzyme activity in specific tumor compartments and patient outcomes. We expect that this modular and multiplexed chemoproteomic platform will be a general approach for drug target engagement, as well as comparative enzyme activity profiling for basic and clinical applications.