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.

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基于蛋白质合成率的D10-亮氨酸测定水生细菌生产力的新方法

摘要：最广泛用于测量细菌产生的方法是tri标记的亮氨酸（³ H-Leu）。尽管此方法提供了方法上的简便性和高灵敏度，但是放射性同位素的使用通常受到法规的限制，特别是在现场环境中。在这项研究中，我们开发了一种非放射性方法，该方法使用氘标记的亮氨酸（（CD ₃）₂ CDCD ₂ CD（NH ₂）COOH; D ₁₀ -Leu）；基于蛋白质的合成速率来测量细菌的生产力。然后将所提出的方法与³ H-Leu方法进行比较和验证。建议方法的步骤是（1）掺入D ₁₀-Leu被细菌感染，（2）酸水解（HCl）变成氨基酸，（3 ）通过液相色谱质谱法（LC-MS / MS）定量D ₁₀ -Leu（m / z 142.10）。在LC-MS / MS分析中，我们检测到的d较大量₉ -Leu（M / Z 141.10）和d ₈ -Leu（M / Z 140.10）比d的₁₀ -Leu，这表明PJ内置d ₁₀ - Leu在脱氨和氨基转移酶反应中迅速代谢。D ₁₀ -Leu，D ₁₀ -Leu + D ₉ -Leu（D ₁₀ + D ₉ -Leu）和D ₁₀ -Leu + D的掺入率₉ -Leu + D ₈ -Leu（D ₁₀ + D ₉ + D ₈ -Leu）与³ H-Leu呈显着正相关，证实了该方法的有效性。由于无法检测到D ₇ -Leu（m / z 139.10），因此可以通过D ₁₀ + D ₉ + D ₈ -Leu测量准确估算掺入蛋白质的外源亮氨酸的量。使用稳定同位素标记的亮氨酸的新的基于化合物的定量方法可以成为估算纯蛋白质合成率以测量细菌产生的有力工具。