Dynamics of Non-Canonical Amino Acid-Labeled Intra- and Extracellular Proteins in the Developing Mouse

• Published in: Cellular and molecular bioengineering Vol. 12; no. 5; pp. 495 - 509
• Main Authors: Saleh, Aya M., Jacobson, Kathryn R., Kinzer-Ursem, Tamara L., Calve, Sarah
• Format: Journal Article
• Language: English
• Published: New York Springer US 01-10-2019; Springer Nature B.V
• Subjects: Alkynes; Amino acids; Assembly; Avidin; Beads; Biological and Medical Physics; Biomaterials; Biomedical Engineering and Bioengineering; Biomedical Engineering/Biotechnology; Biophysics; Biotin; Cell Biology; Contamination; Cytoskeleton; Embryos; Engineering; Extracellular matrix; Gel electrophoresis; Peptide mapping; Protein biosynthesis; Protein synthesis; Protein turnover; Proteins; Proteomes; Sodium lauryl sulfate; Western blotting; BONCAT; Azidohomoalanine; Click chemistry; Diazo biotin-alkyne; Mass spectrometry
• ISSN: 1865-5025; 1865-5033
• DOI: 10.1007/s12195-019-00592-1

Introduction
Mapping protein synthesis and turnover during development will provide insight into functional tissue assembly; however, quantitative
in vivo
characterization has been hindered by a lack of tools. To address this gap, we previously demonstrated murine embryos can be labeled with the non-canonical amino acid azidohomoalanine (Aha), which enables the enrichment and identification of newly synthesized proteins. Using this technique, we now show how protein turnover varies as a function of both time and cellular compartment during murine development.
Methods
Pregnant C57BL/6 mice were injected with Aha or PBS (control) at different embryonic time points. Aha-labeled proteins from homogenized E12.5 and E15.5 embryos were conjugated with diazo biotin-alkyne, bound to NeutrAvidin beads, selectively released, then processed for either SDS-PAGE or LC–MS/MS. For turnover studies, embryos were harvested 0–48 h after Aha injection at E12.5, separated into different cellular fractions based on solubility, and analyzed
via
western blotting.
Results
We developed an enhanced method for isolating Aha-labeled proteins from embryos that minimizes background signal from unlabeled proteins and avidin contamination. Approximately 50% of all identified proteins were found only in Aha samples. Comparing proteins present in both Aha and PBS samples, 90% were > 2-fold enriched in Aha-treated embryos. Furthermore, this method could resolve differences in the Aha-labeled proteome between developmental time points. Newly synthesized Aha-labeled proteins were observed by 3 h and peak labeling was around 6 h. Notably, extracellular matrix and cytoskeletal turnover appeared lower than the cytosolic fraction.
Conclusions
The methods developed in this work enable the identification and quantification of protein synthesis and turnover in different tissue fractions during development. This will provide insight into functional tissue assembly and ultimately inform the design of regenerative therapies that seek to promote growth and repair.