An Integrative Biology Approach to Quantify the Biodistribution of Azidohomoalanine In Vivo.

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.