HPLC Method for Simultaneous Quantification of Marine Microbial Metabolic Stocks and Turnover Rates of ATP, ADP, and AMP

Productivity controls the role of the ocean as a global carbon sink. The energy required for microbial metabolic activity is obtained from light or organic carbon for phototrophs and heterotrophs, respectively. Variations in these metabolic functions can be identified via changes in metabolic energy. The adenylate system includes adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP). These molecules are ubiquitous in microbes, necessary for performing metabolic processes, and required for growth and maintaining life. Primarily, energy is transferred within cells through the production of ATP from inorganic phosphate and ADP. Our ability to measure these energy molecules in marine microbes has been hindered by the throughput and quantitative ability of the analytical techniques used. A firefly lantern extract and luciferase method is widely used for quantification of ATP, but does not allow for the quantification of ADP or AMP. Alternatively, a thin layer chromatography (TLC) method is used to purify ATP, ADP, and AMP, but the process is laborious and restricts the ability to run a large volume of samples, such as with extensive geographic or temporal surveys. High performance liquid chromatography (HPLC) provides a high throughput quantitative method for the purification and analysis of ATP, ADP, and AMP, but previous applications were primarily designed for high biomass plant and animal tissues. We have adapted and optimized these established HPLC methods to quantify ATP, ADP, and AMP in natural marine samples, allowing us to achieve a more exact purification with higher sample throughput than with the previously established methods. Coupling this HPLC purification with radiolabeled phosphate incubations enables ATP, ADP, and AMP turnover rate measurements for marine microbes. Measuring the turnover rate of ATP is a proxy for measuring the turnover rate of energy in the cells, whereas the turnover rates of AMP are correlated with net growth and generation time. We present an HPLC chromatography method to separate and quantify ATP, ADP, and AMP at concentrations as low as 6.4 pmol on column, and its application for determining ATP and AMP turnover rates in natural marine samples, providing a powerful new tool that can be widely deployed for assessing microbial metabolism.