TePhe, a tellurium-containing phenylalanine mimic, allows monitoring of protein synthesis in vivo with mass cytometry

Abstract

Protein synthesis is central to maintaining cellular homeostasis and its study is critical to understanding the function and dysfunction of eukaryotic systems. Here we report L-2-tellurienylalanine (TePhe) as a noncanonical amino acid for direct measurement of protein synthesis. TePhe is synthetically accessible, nontoxic, stable under biological conditions, and the tellurium atom allows its direct detection with mass cytometry, without postexperiment labeling. TePhe labeling is competitive with phenylalanine but not other large and aromatic amino acids, demonstrating its molecular specificity as a phenylalanine mimic; labeling is also abrogated in vitro and in vivo by the protein synthesis inhibitor cycloheximide, validating TePhe as a translation reporter. In vivo, imaging mass cytometry with TePhe visualizes translation dynamics in the mouse gut, brain, and tumor. The strong performance of TePhe as a probe for protein synthesis, coupled with the operational simplicity of its use, suggests TePhe could become a broadly applied molecule for measuring translation in vitro and in vivo. mass cytometry | protein synthesis | tellurium

Validation of l‐Tellurienylalanine as a Phenylalanine Isostere

“…In our previous report, synthesis of l ‐TePhe from Boc‐ l ‐propargylglycine was hampered by acid‐catalyzed degradation during the final Boc‐deprotection step . Here, the synthesis of Fmoc‐TePhe proceeded along a similar route to the previous report except for the use of trityl protection for the α‐amine, which permitted deprotection under milder conditions.…”

“…Noncanonical amino acids (ncAAs) have been used extensively to study and modulate the biochemical and structural properties of proteins; they have also served as important tools for the interrogation of biological phenomena . Our contribution to the growing toolkit of ncAAs is TePhe, a tellurium bearing Phe mimic . We previously demonstrated utility of TePhe as a global protein synthesis reporter.…”

“…Moreover, the isosteric nature of TePhe could be potentially generalized to tellurophenes as phenyl mimics, thus greatly increasing the potential for novel probe generation. The use of TePhe isotopologues, multiplexed with additional MC reagents, will enable time‐resolved experiments and deep‐profiling of biological samples at a cellular level . Ongoing work is also focused on the production of recombinant proteins through metabolic incorporation of TePhe.…”

Methods for analyzing tellurium imaging mass cytometry data

“…In this way, IMC can be used to visualize processes or microenvironments including protein synthesis and cellular hypoxia. [5, 6] Imaging of these compounds is not dependent on antibody-epitope binding and as such represents direct visualization of the probe itself. Tellurium exists naturally as a mixture of eight stable isotopes, of which six are commercially available in an isotopically-enriched form.…”

Methods for Analyzing Imaging Mass Cytometry Data with Tellurium Probes

“…Probes for specific biological processes can be synthesized by linking tellurophenes, which are aromatic, stable, and non-toxic, to activity-based functional groups that covalently bind cells of a certain phenotype. In this way, IMC can be used to visualize processes or microenvironments including protein synthesis and cellular hypoxia (5,6). Imaging of these compounds is not dependent on antibodyepitope binding and as such represents direct visualization of the probe itself.…”