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

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 ¹⁵N₁₀-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.