Saleh Aya M, VanDyk Tyler G, Jacobson Kathryn R, Khan Shaheryar A, Calve Sarah, Kinzer-Ursem Tamara L
Weldon School of Biomedical Engineering, Purdue University, 206 S. Martin Jischke Dr, West Lafayette, IN 47906 USA.
Purdue University Interdisciplinary Life Science Program, 155 S. Grant Street, West Lafayette, IN 47907 USA.
Cell Mol Bioeng. 2023 Mar 23;16(2):99-115. doi: 10.1007/s12195-023-00760-4. eCollection 2023 Apr.
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 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.
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 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.
Our results demonstrate that we can reproducibly predict protein labeling and that the administration of this analog does not significantly alter 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.
The online version contains supplementary material available at 10.1007/s12195-023-00760-4.
新合成蛋白质（NSPs）的鉴定和定量对于理解发育和疾病中的蛋白质动态至关重要。利用内源性翻译机制，通过非天然氨基酸（ncAAs）选择性标记NSPs来探测新生蛋白质组，然后可以用质谱法定量。我们之前已经证明，通过注射叠氮高丙氨酸（Aha，一种ncAA和甲硫氨酸（Met）类似物）标记小鼠蛋白质组是可行的，无需耗尽Met。Aha标记可以解决时间蛋白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。
为了填补这些空白，我们创建了一个确定性的、关于Aha在小鼠体内的动力学转运和掺入的房室模型。模型结果表明能够预测Aha在各种组织和给药模式下的分布以及蛋白质标记情况。为了确定该方法在研究中的适用性，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给药在小鼠中引起的代谢改变最小。
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，这种类似物的给药不会显著改变生理状态。我们期望这个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对刺激反应的实验。
在线版本包含可在10.1007/s12195-023-00760-4获取的补充材料。
