Global Profiling of the Lysine Crotonylome in Different Pluripotent States

Understanding the mechanisms of pluripotency maintenance and exit are important for developmental biology and regenerative medicine. However, studies of pluripotency and post-translational modifications of proteins are scarce. To systematically profile protein crotonylation in mouse pluripotent stem cells (PSCs) in different culture conditions, we used affinity purification of crotonylated peptides, TMT labeling, and LC-MS/MS. Our study included PSCs in ground, metastable, and primed state, as well as metastable PSCs undergoing early pluripotency exit. We have successfully identified 8,102 crotonylation sites in 2,578 proteins, among which 3,628 sites in 1,426 proteins were with high-confidence. These high-confidence crotonylated proteins are enriched for factors involved in functions related to pluripotency such as RNA biogenesis, central carbon metabolism, and proteasome function. Our atlas of protein crotonylation will be valuable for further studies of pluripotency regulation and may also provide insights into the role of crotonylation in other cell fate transitions.

解析多能性维持与退出的机制，对于发育生物学及再生医学领域均具有重要意义。然而，目前针对多能性及蛋白质翻译后修饰的相关研究仍较为匮乏。为系统刻画不同培养条件下小鼠多能干细胞（mouse pluripotent stem cells，PSCs）内的蛋白质巴豆酰化（crotonylation）图谱，本研究采用巴豆酰化肽段亲和纯化、TMT标记及液相色谱-串联质谱（LC-MS/MS）技术。本研究涵盖了处于原始态、亚稳态及始发态的多能干细胞，以及经历早期多能性退出的亚稳态多能干细胞。本研究成功在2578个蛋白质中鉴定出8102个巴豆酰化位点，其中1426个蛋白质的3628个位点具有高置信度。上述高置信度巴豆酰化蛋白质显著富集于多能性相关的功能通路，包括RNA生物发生、中心碳代谢及蛋白酶体功能等。本研究构建的蛋白质巴豆酰化图谱，将为多能性调控的后续研究提供重要参考，同时也可为解析巴豆酰化在其他细胞命运转变过程中的作用提供新视角。