Aya M Saleh, Tyler G VanDyk, Kathryn R Jacobson, Shaheryar A Khan, Sarah Calve, Tamara L Kinzer-Ursem
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引用次数: 0
Abstract
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.
Supplementary information: The online version contains supplementary material available at 10.1007/s12195-023-00760-4.
背景:新合成蛋白质(NSPs)的鉴定和定量对于了解发育和疾病过程中的蛋白质动态至关重要。利用非典型氨基酸(ncAAs)可选择性地标记利用内源翻译机制合成的新蛋白质,然后用质谱法对其进行定量,从而实现对新生蛋白质组的探测。我们之前已经证明,通过注射叠氮高丙氨酸(Aha)(一种 ncAA 和蛋氨酸(Met)的类似物)可以标记体内小鼠蛋白质组,而无需消耗 Met。Aha 标记可以解决蛋白质时间动态显著的生物学问题。然而,要获得这种时间分辨率,需要更全面地了解 Aha 在组织中的分布动力学:为了弥补这些不足,我们建立了一个确定性的小鼠体内 Aha 转运和结合动力学分区模型。模型结果表明,该模型能够预测 Aha 在各种组织和给药模式中的分布和蛋白质标记。为了确定该方法是否适用于体内研究,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 给药对正常生理机能的影响。我们的研究结果表明,服用 Aha 对小鼠的代谢改变极小:我们的研究结果表明,我们可以重复预测蛋白质标记,而且在我们的实验研究过程中,服用这种类似物不会显著改变体内生理学。我们希望这一模型能成为指导未来实验的有用工具,利用这一技术研究蛋白质组对刺激的反应:在线版本包含补充材料,可查阅 10.1007/s12195-023-00760-4。
期刊介绍:
The field of cellular and molecular bioengineering seeks to understand, so that we may ultimately control, the mechanical, chemical, and electrical processes of the cell. A key challenge in improving human health is to understand how cellular behavior arises from molecular-level interactions. CMBE, an official journal of the Biomedical Engineering Society, publishes original research and review papers in the following seven general areas:
Molecular: DNA-protein/RNA-protein interactions, protein folding and function, protein-protein and receptor-ligand interactions, lipids, polysaccharides, molecular motors, and the biophysics of macromolecules that function as therapeutics or engineered matrices, for example.
Cellular: Studies of how cells sense physicochemical events surrounding and within cells, and how cells transduce these events into biological responses. Specific cell processes of interest include cell growth, differentiation, migration, signal transduction, protein secretion and transport, gene expression and regulation, and cell-matrix interactions.
Mechanobiology: The mechanical properties of cells and biomolecules, cellular/molecular force generation and adhesion, the response of cells to their mechanical microenvironment, and mechanotransduction in response to various physical forces such as fluid shear stress.
Nanomedicine: The engineering of nanoparticles for advanced drug delivery and molecular imaging applications, with particular focus on the interaction of such particles with living cells. Also, the application of nanostructured materials to control the behavior of cells and biomolecules.