Separation and Quantification of Cyclic di‐AMP, ‐GMP, and Cyclic GAMP in Bacteria Using LC–MS/MS

ABSTRACT This study summarizes the optimization of a selective method for UHPLC‐separation and subsequent tandem mass spectrometric detection of adenosyl‐ and guanosyl‐containing cyclic dinucleotides in bacteria. Cyclic dinucleotides are a class of bacterial second messenger molecules, and their cellular concentrations are tightly regulated by biosynthesis and enzymatic breakdown. Although bacteria, according to present knowledge, only produce 3′,3′‐linked cyclic dinucleotides, other nucleotide variants also exist, including structural isomers, which may lead to misidentifications. Mixtures of the 2′,2′‐, 2′,3′‐, and 3′,3′‐linked isomers of cyclic di‐AMP, cyclic di‐GMP, and cyclic GAMP were separated using an octadecylsilane‐amide column. Subsequent tandem mass spectrometric detection was based on monitoring the adenosyl‐ or guanosyl‐product ions for quantification, and additional monitoring of up to three product ions for verification. We show the presence of an unidentified putative structural isomer of cyclic di‐AMP in several bacterial species. Cyclic di‐AMP was quantified using an isotope dilution approach and 15 N 10 ‐cyclic di‐AMP as an internal standard, whereas instrument calibration for other variants was performed using matrix‐matched calibration. The combined measurement uncertainty u′ for the quantification of the nine cyclic dinucleotide variants in anion‐exchanged bacterial extracts was 10%–41%, determined at an extract concentration of 12 nM. Our study also demonstrated the application of total protein measurements in resuspended, nucleotide‐extracted, bacterial pellets to normalize nucleotide concentrations.