Pluripotent stem cells (PSCs) can be expanded in vitro in different culture conditions, resulting in a spectrum of cell states with distinct properties. Understanding how PSCs transition from one state to another, ultimately leading to lineage-specific differentiation, is important for developmental biology and regenerative medicine. Although there is significant information regarding gene expression changes controlling these transitions, less is known about post-translational modifications of proteins. Protein crotonylation is a newly discovered post-translational modification where lysine residues are modified with a crotonyl group. Here, we employed affinity purification of crotonylated peptides and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to systematically profile protein crotonylation in mouse PSCs in different states including ground, metastable, and primed states, as well as metastable PSCs undergoing early pluripotency exit. We successfully identified 3628 high-confidence crotonylated sites in 1426 proteins. These crotonylated proteins are enriched for factors involved in functions/processes related to pluripotency such as RNA biogenesis, central carbon metabolism, and proteasome function. Moreover, we found that increasing the cellular levels of crotonyl-coenzyme A (crotonyl-CoA) through crotonic acid treatment promotes proteasome activity in metastable PSCs and delays their differentiation, consistent with previous observations showing that enhanced proteasome activity helps to sustain pluripotency. Our atlas of protein crotonylation will be valuable for further studies of pluripotency regulation and may also provide insights into the role of metabolism in other cell fate transitions.

多能干细胞 (PSC) 可以在不同的培养条件下在体外扩增，从而产生具有不同特性的一系列细胞状态。了解 PSC 如何从一种状态过渡到另一种状态，最终导致谱系特异性分化，对于发育生物学和再生医学很重要。尽管有关于控制这些转变的基因表达变化的重要信息，但对蛋白质的翻译后修饰知之甚少。蛋白质巴豆酰化是一种新发现的翻译后修饰，其中赖氨酸残基被巴豆酰基修饰。在这里，我们采用巴豆酰化肽的亲和纯化和液相色谱-串联质谱法 (LC-MS/MS) 来系统地分析处于不同状态的小鼠 PSC 中的蛋白质巴豆酰化，包括基态、亚稳态和引发状态，以及处于早期阶段的亚稳态 PSC。多能性出口。我们成功地在 1426 种蛋白质中鉴定了 3628 个高度可信的巴豆酰化位点。这些巴豆酰化蛋白富含与多能性相关的功能/过程的因子，如 RNA 生物发生、中心碳代谢和蛋白酶体。功能。此外，我们发现通过巴豆酸处理增加巴豆酰辅酶 A（巴豆酰辅酶 A）的细胞水平可促进亚稳态 PSC 中的蛋白酶体活性并延迟其分化，这与之前的观察结果一致，表明增强的蛋白酶体活性有助于维持多能性。我们的蛋白质巴豆酰化图谱对于进一步研究多能性调控很有价值，并且还可以提供对代谢在其他细胞命运转变中的作用的见解。