Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 6, 32377

Incorporation of Non-Canonical Amino Acids Into the Developing Murine Proteome

Affiliations

Incorporation of Non-Canonical Amino Acids Into the Developing Murine Proteome

Sarah Calve et al. Sci Rep.

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.

Figures

Figure 1
Figure 1. Metabolic labeling with non-canonical amino acids.
(A) Amino acids in use in this study: Methionine (Met), Azidohomoalanine (AHA), Homopropargylglycine (HPG). (B) Peptides and proteins that naturally contain Met will instead be synthesized with AHA or HPG in the place of Met. (C) Newly synthesized proteins that have incorporated HPG (blue ribbon) are labeled with a fluorophore-conjugated azide (red star). Copper-catalyzed azide/alkyne cycloaddition results in a stable triazole adduct. (D) Mice were injected with varying amounts of ncAA for two days. Tissues were harvested, solubilized, reacted with azide- or alkyne-conjugated fluorophore then analyzed with SDS-PAGE.
Figure 2
Figure 2. Systemic and dose-dependent incorporation of non-canonical amino acids (ncAA) in murine tissues.
(A) Juvenile mice were injected IP with 0.1 mg/g per day with AHA, HPG or PBS for two days and heart (H), lung (L), brain (B), skeletal muscle (M) and kidney (K) lysates were compared. (B) Brain lysates from juvenile mice injected with 0.025–0.1 mg/g AHA, HPG or PBS. Tissue lysates were labeled with fluorophore-conjugated azide or alkyne, resolved using SDS-PAGE, fluorescently imaged then stained for total protein with Coomassie Blue to confirm equal loading. Representative results from N ≥ 3 independent experiments.
Figure 3
Figure 3. AHA is robustly incorporated into the developing murine proteome.
(A) Dams injected with 0.1 mg/g per day for 2 days incorporated AHA into the developing embryos and their own tissues; however, the latter was much lower as indicated by the need for contrast enhancement (L = lung). Tissue lysates were labeled with fluorophore-conjugated alkyne, resolved using SDS-PAGE, fluorescently imaged then stained for total protein with Coomassie Blue to confirm equal loading. Representative results from N ≥ 3 independent experiments. Arrows indicate the bands excised for proteomics analysis described in Fig. 4. (B) Age matched embryos from control and AHA-injected dams show no difference in size and protein banding pattern (A). (C) Intensity tracings of the total protein bands in lanes 2 (AHA) and 3 (PBS) show that overall protein expression was not changed by AHA administration. Fluorescence intensity of proteins in lane 2 (dotted line) reveals that peaks occur in the same locations as in the total protein (solid lines). Representative results from N ≥ 3 pregnancies.
Figure 4
Figure 4. AHA administration minimally impacts the embryonic proteome.
Two bands at ~42kDa and 48kDa with high levels of AHA labeling (arrows Fig. 3A) were digested in-gel and analyzed using LC-MS/MS. (A) The majority of proteins present in AHA and PBS samples were identified in both populations (top, Table S1). Of the proteins expressed in both AHA and PBS, only 5–10% showed significantly different levels of expression as analyzed using MaxQuant (p < 0.05, bottom). (B) Heat map indicating the identities and log2-fold difference in expression of all proteins differentially expressed in bands 1 and 2.

Similar articles

See all similar articles

Cited by 20 PubMed Central articles

See all "Cited by" articles

References

    1. Choudhary C. & Mann M. Decoding signalling networks by mass spectrometry-based proteomics. Nat Rev Mol Cell Biol 11, 427–439 (2010). - PubMed
    1. Sun L. et al. . Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development. Sci Rep 4, 4365 (2014). - PMC - PubMed
    1. Peshkin L. et al. . On the Relationship of Protein and mRNA Dynamics in Vertebrate Embryonic Development. Dev Cell 35, 383–394 (2015). - PMC - PubMed
    1. Hartl D. et al. . Transcriptome and proteome analysis of early embryonic mouse brain development. Proteomics 8, 1257–1265 (2008). - PubMed
    1. Lucitt M. B. et al. . Analysis of the zebrafish proteome during embryonic development. Mol Cell Proteomics 7, 981–994 (2008). - PMC - PubMed

Publication types

Feedback