Real-Time Mass Spectrometry Measurements of Respiration Rates in Biological Systems

Many organisms process carbon and other nutrients to generate energy through aerobic respiration where organic carbon compounds are broken down and oxygen is consumed, producing carbon dioxide and water. Respiration is indicative of active metabolism, and respiration rates are proportional to the amount of living biomass in an ecosystem. Although there are many methods for measuring respiration rates in the laboratory, current systems, such as infrared gas analyzers, are limited in their ability to independently resolve isotopomer fluxes across a range of relevant gases including both CO₂ and O₂ in real-time. Therefore, monitoring of biological respiration in real time under controlled laboratory conditions would enable better understanding of cellular physiology. To address this challenge, we developed a real time mass spectrometry (RTMS) manifold that simultaneously measures production and consumption of multiple gases and their isotopologues in seconds with the speed and sensitivity necessary to characterize rapidly changing respiration events as they occur. This universal manifold can be fitted to a variety of instruments and affords the same analytical precision and accuracy of the instrument while allowing for the real time measurements. Here, we paired the manifold to a single quad MS with an electron impact (EI) source operated in scan mode to detect extracted target gases by their respective masses (e.g., ¹²CO₂ at mass 44, ¹³CO₂ at 45). We demonstrated applicability of the RTMS instrument to different biological ecosystems (bacterial cultures, plants, and soil), and in all cases, we were able to detect simultaneous and rapid measurements of multiple gases in real time, providing novel insights into complex respiratory metabolism and the influence of biological and environmental factors.