Protein synthesis rates derived from incorporation of non-radioactively labeled phenylalanine in Antarctic fish

Temperature dependent growth is an important indicator to understand the thermal tolerance of organisms and to project their vulnerability to future climate change. Direct measurements of temperature dependent weight gains, however, are experimentally challenging and time consuming in long-lived species. Here, we reassess methodology to quantify the in vivo protein synthesis rate from amino acids, as a key component of growth. We developed an analytical method that is both robust against analytical errors and does not require hazardous radioactive materials. We utilized the incorporation of isotopically 13C15N-labeled-phenylalanine into fish muscle followed by quantification by liquid chromatography mass spectrometry to calculate accurate net protein synthesis rates in muscle tissue of Antarctic fish, Pachycara brachycephalum, in vivo. Specifically, we injected 150 mM of 13C9H915N1 phenylalanine intraperitoneally and sampled muscle tissue in 1,5h steps between 0 and 6 hours after injection. We quantified labeled and unlabeled phenylalanine both in muscle protein and in the cytosol. This allowed us to critically re-evaluate three different protein synthesis rate (Ks) calculation methodologies that have been developed over the last decades. The calculated values differ by more than 70-fold (0.048 ± 0.021% day-1 up to 3.56 ± 2.16 day-1) between methods. We argue that the Ks calculation including a proportionate ratio of protein synthesis from (unlabeled) free amino acids yields the most realistic Ks values for cold water fish. Eventually, the standardization of the net protein synthesis rate calculation will lead to dependable quantitative representations of organismal stress in response to climate change.