Acyl-coenzyme A (CoA)/protein interactions are essential for life. Despite this importance, their global scope and selectivity remains undefined. Here, we describe CATNIP (CoA/AcetylTraNsferase Interaction Profiling), a chemoproteomic platform for the high-throughput analysis of acyl-CoA/protein interactions in endogenous proteomes. First, we apply CATNIP to identify acetyl-CoA-binding proteins through unbiased clustering of competitive dose-response data. Next, we use this method to profile the selectivity of acyl-CoA/protein interactions, leading to the identification of specific acyl-CoA engagement signatures. Finally, we apply systems-level analyses to assess the features of protein networks that may interact with acyl-CoAs, and use a strategy for high-confidence proteomic annotation of acetyl-CoA-binding proteins to identify a site of non-enzymatic acylation in the NAT10 acetyltransferase domain that is likely driven by acyl-CoA binding. Overall, our studies illustrate how chemoproteomics and systems biology can be integrated to understand the roles of acyl-CoA metabolism in biology and disease.

酰基辅酶 A (CoA)/蛋白质相互作用对于生命至关重要。尽管如此重要，但它们的全球范围和选择性仍然不确定。在这里，我们描述了 CATNIP（CoA/AcetylTraNsferase Interaction Profiling），这是一种化学蛋白质组学平台，用于内源蛋白质组中酰基辅酶 A/蛋白质相互作用的高通量分析。首先，我们应用 CATNIP 通过竞争性剂量反应数据的无偏聚类来识别乙酰辅酶A结合蛋白。接下来，我们使用这种方法来分析酰基辅酶A/蛋白质相互作用的选择性，从而识别特定的酰基辅酶A接合特征。最后，我们应用系统级分析来评估可能与酰基辅酶A相互作用的蛋白质网络的特征，并使用乙酰辅酶A结合蛋白的高置信度蛋白质组注释策略来识别非酶酰化位点NAT10 乙酰转移酶结构域可能由酰基辅酶 A 结合驱动。总的来说，我们的研究说明了如何整合化学蛋白质组学和系统生物学来了解酰基辅酶A代谢在生物学和疾病中的作用。