Examining targeted protein degradation from physiological and analytical perspectives: Enabling translation between cells and subjects.
The ability to target specific proteins for degradation may open a new door toward developing therapeutics. Although effort in chemistry is essential for advancing this modality, i.e., one needs to generate proteolysis targeting chimeras (bifunctional molecules, also referred to as PROTACS) or "molecular glues" to accelerate protein degradation, we suspect that investigations could also benefit by directing attention toward physiological regulation surrounding protein homeostasis, including the methods that can be used to examine changes in protein kinetics. This perspective will first consider some metabolic scenarios that might be of importance when one aims to change protein abundance by increasing protein degradation. Specifically, could protein turnover impact the apparent outcome? We will then outline how to study protein dynamics by coupling stable isotope tracer methods with mass spectrometry-based detection; since the experimental conditions could have a dramatic effect on protein turnover, special attention is directed toward the application of methods for quantifying protein kinetics using in vitro and in vivo models. Our goal is to present key concepts that should enable mechanistically informed studies which test targeted protein degradation strategies.
Daurio NA;Zhou H;Chen Y;Sheth PR;Imbriglio JE;McLaren DG;Tawa P;Rachdaoui N;Previs MJ;Kasumov T;O'Neil J;Previs SF
ACS Chemical Biology
2020
2020-09-30
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
journalArticle
<a href="http://doi.org/10.1021/acschembio.0c00380" target="_blank" rel="noreferrer noopener">10.1021/acschembio.0c00380</a>
Isotope Fractionation during Gas Chromatography Can Enhance Mass Spectrometry-Based Measures of (2)H-Labeling of Small Molecules.
stable isotopes; data integration; gas chromatography-mass spectrometry; isotope fractionation; metabolic flux; Savitzky-Golay
Stable isotope tracers can be used to quantify the activity of metabolic pathways. Specifically, (2)H-water is quite versatile, and its incorporation into various products can enable measurements of carbohydrate, lipid, protein and nucleic acid kinetics. However, since there are limits on how much (2)H-water can be administered and since some metabolic processes may be slow, it is possible that one may be challenged with measuring small changes in isotopic enrichment. We demonstrate an advantage of the isotope fractionation that occurs during gas chromatography, namely, setting tightly bounded integration regions yields a powerful approach for determining isotope ratios. We determined how the degree of isotope fractionation, chromatographic peak width and mass spectrometer dwell time can increase the apparent isotope labeling. Relatively simple changes in the logic surrounding data acquisition and processing can enhance gas chromatography-mass spectrometry measures of low levels of (2)H-labeling, this is especially useful when asymmetrical peaks are recorded at low signal:background. Although we have largely focused attention on alanine (which is of interest in studies of protein synthesis), it should be possible to extend the concepts to other analytes and/or hardware configurations.
Downes DP; Kasumov T; Daurio NA; Wood NB; Previs MJ; Sheth PR; McLaren DG; Previs SF
Metabolites
2020
2020-11-20
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
journalArticle
<a href="http://doi.org/10.3390/metabo10110474" target="_blank" rel="noreferrer noopener">10.3390/metabo10110474</a>