Dynamics of Non-Canonical Amino Acid-Labeled Intra- and Extracellular Proteins in the Developing Mouse
Dynamics of Non-Canonical Amino Acid-Labeled Intra- and Extracellular Proteins in the Developing Mouse
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发育中小鼠中非规范氨基酸标记的细胞内和细胞外蛋白质的动态
DOI：
10.1007/s12195-019-00592-1
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发表时间：
2019
影响因子：
2.8
通讯作者：
Calve, Sarah
中科院分区：
文献类型：
--
作者：
Saleh, Aya M.;Jacobson, Kathryn R.;Kinzer-Ursem, Tamara L.;Calve, Sarah
在发育过程中绘制蛋白质合成和周转图将有助于了解功能性组织组装；然而，由于缺乏工具，定量体内表征一直受到阻碍。为了解决这一空白，我们先前证明小鼠胚胎可以用非规范氨基酸叠氮同质丙氨酸（Aha）进行标记，这使得新合成的蛋白质能够富集和鉴定。利用这种技术，我们现在展示了在小鼠发育过程中，蛋白质周转如何随着时间和细胞间隔的变化而变化。方法C57BL/6妊娠小鼠在不同胚胎时间点注射Aha或PBS（对照）。从匀浆的E12.5和E15.5胚胎中，将aha标记的蛋白与重氮生物素-炔偶联，结合到NeutrAvidin珠上，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 h收集胚胎，根据溶解度将其分离成不同的细胞组分，并通过western blotting进行分析。结果我们开发了一种从胚胎中分离aha标记蛋白的增强方法，该方法可以最大限度地减少未标记蛋白和亲和素污染的背景信号。大约50%的鉴定蛋白仅在Aha样品中发现。比较Aha和PBS样品中存在的蛋白质，在Aha处理的胚胎中，90%的蛋白质富集了10倍。此外，该方法可以解决不同发育时间点aha标记蛋白质组的差异。3 h观察新合成的aha标记蛋白，6 h左右出现标记峰。值得注意的是，细胞外基质和细胞骨架的周转率明显低于胞质部分。结论所建立的方法可用于鉴定和定量发育过程中不同组织组分的蛋白质合成和转换。这将提供对功能性组织组装的深入了解，并最终为寻求促进生长和修复的再生疗法的设计提供信息。
IntroductionMapping protein synthesis and turnover during development will provide insight into functional tissue assembly; however, quantitativein vivocharacterization 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.MethodsPregnant 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 analyzedviawestern blotting.ResultsWe 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.ConclusionsThe 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.
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