This work introduces light-activated bioorthogonal noncanonical amino acid tagging (laBONCAT) as a method to selectively label, isolate, and identify proteins newly synthesized at user-defined regions in tissue culture. By photocaging l-azidohomoalanine (Aha), metabolic incorporation into proteins is prevented. The caged compound remains stable for many hours in culture, but can be photochemically liberated rapidly and on demand with spatial control. Upon directed light exposure, the uncaged amino acid is available for local translation, enabling downstream proteomic interrogation via bioorthogonal conjugation. Exploiting the reactive azide moiety present on Aha’s amino acid side chain, we demonstrate that newly synthesized proteins can be purified for quantitative proteomics or visualized in synthetic tissues with a new level of spatiotemporal control. Shedding light on when and where proteins are translated within living samples, we anticipate that laBONCAT will aid in understanding the progression of complex protein-related disorders.

中文翻译：

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通过光笼生物正交非典型氨基酸的光激活蛋白质组学标记。

这项工作引入了光激活生物正交非规范氨基酸标签（laBONCAT），作为一种选择性标记，分离和识别在组织培养中用户定义区域新合成的蛋白质的方法。通过photocaging升-azidohomoalanine（AHA），代谢掺入蛋白质被防止。笼状化合物在培养中可保持稳定数小时，但可以快速进行光化学释放，并可以根据需要通过空间控制将其释放。定向光照后，未封端的氨基酸可用于局部翻译，从而可通过生物正交共轭。利用存在于Aha氨基酸侧链上的反应性叠氮化物部分，我们证明了新合成的蛋白质可以纯化用于定量蛋白质组学或在合成组织中以新的时空控制水平可视化。我们着眼于在活体样品中何时何地翻译蛋白质，我们预计laBONCAT将有助于理解复杂的蛋白质相关疾病的进展。