Salt-Tolerant Metabolomics for Exometabolomic Measurements of Marine Bacterial Isolates.

Abstract

Identifying and quantifying metabolites secreted by microbial isolates can aid in understanding the physiological traits of diverse species and their interaction with the environment. Mass spectrometry-based metabolomics has potential to provide a holistic view of the exometabolism of marine isolates, but the high salt content of such samples interferes with chromatography and ionization during the measurement of polar exometabolites. The most common desalting methods are faced with major limitations, including limited separation of small polar metabolites from salts, the use of organic solvents that cannot accommodate large salt quantities, and sample throughput. Here, we utilize a cyano stationary phase to develop a high-throughput, isocratic liquid chromatography-mass spectrometry (LC-MS) desalting method that mitigates these shortcomings. We demonstrate that counterions present in a common marine growth medium experience distinct elution times, which prevents their coelution with 73 physiologically relevant polar metabolites, effectively minimizing the effects of salt content on ion suppression. We determined optimal salt concentrations for quadrupole time-of-flight (QTOF) MS measurements and limits of quantification in the low micromolar range in the salty matrix. The efficacy of this method was demonstrated through the measurement of exometabolites secreted by three marine bacterial isolates originating from a carrageenan degrading microbial community. This method provides a simple, versatile desalting method for measuring exometabolites of environmental isolates and other biological matrices.