Introduction

Mapping protein synthesis and turnover during development will provide insight into functional tissue assembly; however, quantitative in vivo characterization has been hindered by a lack of tools. To address this gap, we previously demonstrated murine embryos can be labeled with the non-canonical amino acid azidohomoalanine (Aha), which enables the enrichment and identification of newly synthesized proteins. Using this technique, we now show how protein turnover varies as a function of both time and cellular compartment during murine development.

Methods

Pregnant C57BL/6 mice were injected with Aha or PBS (control) at different embryonic time points. Aha-labeled proteins from homogenized E12.5 and E15.5 embryos were conjugated with diazo biotin-alkyne, bound to NeutrAvidin beads, selectively released, then processed for either SDS-PAGE or LC–MS/MS. For turnover studies, embryos were harvested 0–48 h after Aha injection at E12.5, separated into different cellular fractions based on solubility, and analyzed via western blotting.

Results

We developed an enhanced method for isolating Aha-labeled proteins from embryos that minimizes background signal from unlabeled proteins and avidin contamination. Approximately 50% of all identified proteins were found only in Aha samples. Comparing proteins present in both Aha and PBS samples, 90% were > 2-fold enriched in Aha-treated embryos. Furthermore, this method could resolve differences in the Aha-labeled proteome between developmental time points. Newly synthesized Aha-labeled proteins were observed by 3 h and peak labeling was around 6 h. Notably, extracellular matrix and cytoskeletal turnover appeared lower than the cytosolic fraction.

Conclusions

The methods developed in this work enable the identification and quantification of protein synthesis and turnover in different tissue fractions during development. This will provide insight into functional tissue assembly and ultimately inform the design of regenerative therapies that seek to promote growth and repair.

中文翻译：

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发育中小鼠的非经典氨基酸标记的细胞内和细胞外蛋白的动力学。

介绍

在开发过程中绘制蛋白质合成和周转图将提供对功能性组织组装的深入了解；然而，由于缺乏工具，定量的体内表征受到了阻碍。为了解决这一差距，我们之前证明了小鼠胚胎可以用非经典氨基酸叠氮高丙氨酸 (Aha) 进行标记，从而可以富集和鉴定新合成的蛋白质。使用这种技术，我们现在展示了在小鼠发育过程中蛋白质周转如何随时间和细胞区室的变化而变化。

方法

在不同的胚胎时间点给怀孕的 C57BL/6 小鼠注射 Aha 或 PBS（对照）。来自均质化的 E12.5 和 E15.5 胚胎的 Aha 标记蛋白与重氮生物素-炔烃结合，与 NeutrAvidin 珠结合，选择性释放，然后进行 SDS-PAGE 或 LC-MS/MS 处理。对于周转研究，在 E12.5 注射 Aha 后 0-48 小时收获胚胎，根据溶解度分成不同的细胞部分，并通过蛋白质印迹分析。

结果

我们开发了一种增强的方法，用于从胚胎中分离 Aha 标记的蛋白质，从而最大限度地减少来自未标记蛋白质和抗生物素蛋白污染的背景信号。大约 50% 的已鉴定蛋白质仅在 Aha 样本中发现。比较 Aha 和 PBS 样品中存在的蛋白质，90% 在 Aha 处理的胚胎中富集 > 2 倍。此外，这种方法可以解决发育时间点之间 Aha 标记的蛋白质组的差异。新合成的 Aha 标记蛋白在 3 小时内观察到，峰值标记在 6 小时左右。值得注意的是，细胞外基质和细胞骨架转换似乎低于细胞溶质部分。

结论

在这项工作中开发的方法能够在开发过程中识别和量化不同组织部分中的蛋白质合成和周转。这将提供对功能性组织组装的深入了解，并最终为旨在促进生长和修复的再生疗法的设计提供信息。