AME 85:121-129 (2020)  -  DOI: https://doi.org/10.3354/ame01945

ABSTRACT: The most widely used method for measuring bacterial production is tritium-labeled leucine (³H-Leu). Although this method provides methodological simplicity and high sensitivity, the employment of radioactive isotopes is often restricted by regulations, particularly in field settings. In this study, we developed a non-radioactive method for measuring bacterial productivity based on the protein synthesis rate, using deuterium-labeled leucine ((CD₃)₂CDCD₂CD(NH₂)COOH; D₁₀-Leu); the proposed method was then compared and verified with the ³H-Leu method. The procedures of the proposed method are (1) incorporation of D₁₀-Leu by bacteria, (2) acid hydrolysis (HCl) to amino acids and (3) quantification of D₁₀-Leu (m/z 142.10) by liquid chromatography mass spectrometry (LC-MS/MS). In the LC-MS/MS analysis, we detected a larger amount of D₉-Leu (m/z 141.10) and D₈-Leu (m/z 140.10) than that of D₁₀-Leu, suggesting that incorporated D₁₀-Leu was rapidly metabolized such as in deamination and aminotransferase reactions. The incorporation rates of D₁₀-Leu, D₁₀-Leu + D₉-Leu (D₁₀+D₉-Leu) and D₁₀-Leu + D₉-Leu + D₈-Leu (D₁₀+D₉+D₈-Leu) were significantly positively correlated to that of ³H-Leu, confirming the validity of the proposed method. Since D₇-Leu (m/z 139.10) could not be detected, the amount of exogenous leucine incorporated into protein can be accurately estimated through D₁₀+D₉+D₈-Leu measurement. The new compound-based quantification method using stable isotope-labeled leucine can be a powerful tool to estimate pure protein synthesis rate for measuring bacterial production.

KEY WORDS: Bacterial production · Protein synthesis · Leucine · Deoxyadenosine · Stable isotopes · Liquid chromatography mass spectrometry