Incorporation of non-canonical amino acids into the developing murine proteome

Abstract

Analysis of the developing proteome has been complicated by a lack of tools that can be easily employed to label and identify newly synthesized proteins within complex biological mixtures. Here, we demonstrate that the methionine analogs azidohomoalanine and homopropargylglycine can be globally incorporated into the proteome of mice through facile intraperitoneal injections. These analogs contain bio-orthogonal chemical handles to which fluorescent tags can be conjugated to identify newly synthesized proteins. We show these non-canonical amino acids are incorporated into various tissues in juvenile mice and in a concentration dependent manner. Furthermore, administration of these methionine analogs to pregnant dams during a critical stage of murine development, E10.5–12.5 when many tissues are assembling, does not overtly disrupt development as assessed by proteomic analysis and normal parturition and growth of pups. This successful demonstration that non-canonical amino acids can be directly administered in vivo will enable future studies that seek to characterize the murine proteome during growth, disease and repair.

High fidelity visualization of cell-to-cell variation and temporal dynamics in nascent extracellular matrix formation

“…meniscus destabilization) or joint development could lend insight into how matrix proteins form and reorganize during these processes. Recent studies indicate that HPG and AHA can incorporate into developing murine embryos33, supporting the potential scalability of FUNCAT analysis to such model systems.…”

“…This investigation of the biosynthetic response to injury, disease, or altered genetic program could be performed either in vitro (as we assessed tissue engineered construct formation here) or in vivo . Previous studies have examined protein synthesis in drosophila and zebrafish systems and mice141533; scaling FUNCAT to mammalian systems with in vivo models of cartilage degeneration (e.g. meniscus destabilization) or joint development could lend insight into how matrix proteins form and reorganize during these processes.…”

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

“…[77] Contrarily to Eth, Aha is not toxic in mammalian cell lines, neither in whole organisms for short incubation times, and it has been used to selectively label the newly synthesized proteome via BONCAT. [57,79] These results provided a starting point for the differential reassignment of the N-terminus to Aha using the natural substrate preference of EcMetRS, while globally reassigning the internal AUG codons with Eth using the putative orthogonal SaMetRS / SatRNA Met pair.…”

Exploring ribosome composition and newly synthesized proteins through proteomics and potential biomedical applications

“…The attempts evolved from analysis of protein changes in various cells [32] to the alteration of newly synthesized proteomes in organs [33–34] and whole animals [35]. One of the major challenges accompanying this identification process is dealing with low abundances of newly synthesized proteins and their recognition and distinction from the pre-existing protein pool.…”

“…AHA and HPG labeling via intraperitoneal injection was recently used to characterize the proteome in developing mice [34]. First, it was demonstrated that in juvenile mice (25–35 days old), AHA and HPG incorporated into all tissues under investigation (heart, lung, brain, skeletal muscle and kidney) in dosage-dependent increase (0.025, 0.05 and 0.1 mg/g of AHA or HPG per day; 2 days of administration).…”