(287f) Incorporation of Non-Canonical Amino Acids into the Developing Murine Proteome

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 and affinity tags can be conjugated to facilitate identification of newly synthesized proteins within specific time windows during development.

The methionine (Met) analog azidohomoalanine (AHA) carries an azide functional group and is incorporated into sites normally occupied by Met during protein synthesis. Similarly, the Met analog homopropargylglycine (HPG) carries an alkyne functional group and is also incorporated at Met sites during protein synthesis. Both azide and alkyne handles can be covalently conjugated to affinity tags or fluorphores via copper-catalyzed azide-alkyne cycloaddition (CuAAC), a widely used â??clickâ? chemistry reaction. This enables fluorescent detection of the AHA and HPG containing proteins via SDS-PAGE size-separation and enrichment of the AHA and HPG containing proteins for downstream mass spectrometry identification from complex mixtures such as soluble cell lysate or solubilized membrane fractions and extracellular matrix.

 In this work we show these non-canonical amino acids are incorporated into various tissues in juvenile mice and in a concentration dependent manner. Additionally, administration of these methionine analogs to pregnant dams during a critical stage of murine development, E10.5 â?? E12.5 when many tissues are assembling, leads to robust incorporation of AHA and HPG into the embryo and does not overtly disrupt development as assessed by proteomic analysis and normal parturition and growth of pups. Fluorescence imaging as well as intensity profiles of fluorescently labeled cellular fractions from AHA and HPG treated mice reveal unique banding in multiple tissues and robust incorporation of non-canonical amino acids in embryos. Furthermore, fluorescence imaging reveals unique banding in cytoplasmic, nuclear, membrane, and extracellular matrix fractions of developing tissues, suggesting AHA is incorporated into newly synthesized proteins of all types. Comparison of the intensity profiles of the total protein content of AHA and control samples indicates that protein expression was not noticeably altered as a result of AHA administration.

Proteomic analysis of control and AHA-treated embryos showed that less than 10% of identified proteins have significantly different levels of expression (p > 0.05). When considering a more stringent threshold (p > 0.01), the number of differentially expressed proteins dropped significantly to less than 3%. One differentially expressed protein, Pdia6, a member of the protein disulfide isomerase family, is expressed during times of ER stress and is thought to help attenuate the unfolded protein response via mechanisms independent of disulfide bond forming activity. Overall, it is not unexpected that components involved in protein folding show a difference in expression between the two treatments, given the differences in side chain chemistry between Met and AHA. It is important to note that overall AHA treatment has little effect on the identity and quantity of proteins. This corroborates our observation that AHA treatment does not overtly disrupt embryogenesis or subsequent development after parturition. This successful demonstration that AHA and HPG can be directly administered in vivo for labeling of newly synthesized proteins will enable innumerous future studies that seek to characterize protein dynamics during growth, disease and repair.