Background

Identification and quantitation of newly synthesized proteins (NSPs) are critical to understanding protein dynamics in development and disease. Probing the nascent proteome can be achieved using non-canonical amino acids (ncAAs) to selectively label the NSPs utilizing endogenous translation machinery, which can then be quantitated with mass spectrometry. We have previously demonstrated that labeling the in vivo murine proteome is feasible via injection of azidohomoalanine (Aha), an ncAA and methionine (Met) analog, without the need for Met depletion. Aha labeling can address biological questions wherein temporal protein dynamics are significant. However, accessing this temporal resolution requires a more complete understanding of Aha distribution kinetics in tissues.

Results

To address these gaps, we created a deterministic, compartmental model of the kinetic transport and incorporation of Aha in mice. Model results demonstrate the ability to predict Aha distribution and protein labeling in a variety of tissues and dosing paradigms. To establish the suitability of the method for in vivo studies, we investigated the impact of Aha administration on normal physiology by analyzing plasma and liver metabolomes following various Aha dosing regimens. We show that Aha administration induces minimal metabolic alterations in mice.

Conclusions

Our results demonstrate that we can reproducibly predict protein labeling and that the administration of this analog does not significantly alter in vivo physiology over the course of our experimental study. We expect this model to be a useful tool to guide future experiments utilizing this technique to study proteomic responses to stimuli.

中文翻译：

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一种综合生物学方法来量化 Azidohomoalanine 在体内的生物分布

背景

新合成蛋白质 (NSP) 的鉴定和定量对于了解发育和疾病中的蛋白质动力学至关重要。可以使用非规范氨基酸 (ncAAs) 来探测新生的蛋白质组，以利用内源翻译机制选择性地标记 NSP，然后可以用质谱法对其进行定量。我们之前已经证明，通过注射 azidohomoalanine (Aha)、一种 ncAA 和甲硫氨酸 (Met) 类似物，无需 Met 耗尽即可标记体内鼠类蛋白质组。Aha 标记可以解决其中时间蛋白质动力学很重要的生物学问题。然而，获得这种时间分辨率需要更全面地了解组织中的 Aha 分布动力学。

结果

为了解决这些差距，我们创建了 Aha 在小鼠体内的动力学传输和掺入的确定性隔室模型。模型结果表明能够预测各种组织和剂量范例中的 Aha 分布和蛋白质标记。为了确定该方法对体内研究的适用性，我们通过分析各种 Aha 给药方案后的血浆和肝脏代谢组，研究了 Aha 给药对正常生理的影响。我们表明，Aha 管理在小鼠中引起最小的代谢改变。

结论

我们的结果表明，我们可以可重复地预测蛋白质标记，并且在我们的实验研究过程中，这种类似物的施用不会显着改变体内生理学。我们希望该模型成为一个有用的工具，以指导未来利用该技术研究蛋白质组学对刺激的反应的实验。