CORP: The use of deuterated water for the measurement of protein synthesis

Abstract

The use of deuterium oxide (D2O) has greatly expanded the scope of what is possible for the measurement of protein synthesis. The greatest asset of D2O labeling is that it facilitates the measurement of synthesis rates over prolonged periods of time from single proteins through integrated tissue-based measurements. Because the ease of administration, the method is amenable for use in a variety of models and conditions. Although the method adheres to the same rules as other isotope methods, the flexibility can create conditions that are not the same as other approaches and thus requires careful execution to maintain validity and reliability. For this CORP article, we provide a history that gave rise to the method and discuss the advantages and disadvantages of the method, the critical assumptions, guidelines, and best practices based on instrumentation, models, and experimental design. The goal of this CORP article is to propagate additional use of D2O in a manner that produces reliable and valid data.

A Novel Stable Isotope Approach Demonstrates Surprising Degree of Age-Related Decline in Skeletal Muscle Collagen Proteostasis

“…Protein synthesis was determined using our established methods. 28 , 34 Briefly, ∼30–50 mg of skeletal muscle tissue was homogenized 1:20 in isolation buffer (100 m m KCl, 40 m m Tris–HCl, 10 m m Tris Base, 5 m m MgCl 2 , 1 m m EDTA, 1 m m ATP, pH = 7.6) with phosphatase and protease inhibitors (HALT, Thermo Fisher Scientific) using a bead homogenizer (Next Advance Inc., Averill Park, NY, USA). After homogenization, subcellular fractions were isolated via differential centrifugation to obtain myofibrillar and collagen protein fractions.…”

“…The use of the stable isotope deuterium oxide (D 2 O) has facilitated the capture of slowly turning over proteins because it lends itself to long-term (days to weeks) labeling. 28 The assumptions, limitations, and technical application of the method have been detailed to ensure valid results. 28 When calculating synthesis rates from a single time point, standard tracer methodology assumes that the product undergoes complete renewal (ie, turnover).…”

“… 28 The assumptions, limitations, and technical application of the method have been detailed to ensure valid results. 28 When calculating synthesis rates from a single time point, standard tracer methodology assumes that the product undergoes complete renewal (ie, turnover). 29 However, this assumption of complete renewal may not be correct for some proteins, like collagen, that can become resistant to turnover during aging.…”

“… 30 Not accounting for a potential change in the size of the protein pool that is undergoing turnover, such as collagen that becomes resistant to turnover, could impact the interpretation of the resultant data. 28 , 29 Since mass spectrometry analysis by itself cannot determine which proteins are resistant to turnover, alternative strategies are needed.…”

Low amounts of heavy water increase the phase separation propensity of a fragment of the androgen receptor activation domain

“…Indeed, it has been recently shown that percentages of 1,6‐hexanediol commonly used to disrupt LLPS impact enzymatic activity, for example, inactivating kinases and phosphatases 36 . It is therefore necessary to develop new probes to selectively change the stability of condensates: since moderate (up to 70%) mole fractions of D 2 O can be tolerated by live cells, 37 for example in studies of the rate of protein synthesis in physiology, 38 analysis of how additions of this co‐solvent influence apparent phase separation phenomena in cells may represent a useful tool in this field.…”