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

Area of Science:

• Bacteriology
• Analytical Chemistry
• Molecular Biology

Background:

• Cyclic dinucleotides are vital bacterial second messengers regulating cellular processes.
• Accurate identification is crucial as structural isomers can be misidentified.
• Existing methods may struggle to differentiate between various cyclic dinucleotide isomers.

Purpose of the Study:

• To optimize a selective method for UHPLC-separation and MS/MS detection of cyclic dinucleotides in bacteria.
• To differentiate between 2',2'-, 2',3'-, and 3',3'-linked isomers of cyclic di-AMP, cyclic di-GMP, and cyclic GAMP.
• To identify and quantify unknown cyclic dinucleotide variants in bacterial species.

Main Methods:

• Utilized UHPLC with an octadecylsilane-amide column for isomer separation.
• Employed tandem mass spectrometry (MS/MS) with targeted ion monitoring for quantification and verification.
• Applied isotope dilution and matrix-matched calibration for accurate quantification of cyclic di-AMP and other variants.

Main Results:

• Successfully separated and detected nine cyclic dinucleotide variants, including structural isomers.
• Identified an unknown putative structural isomer of cyclic di-AMP in several bacterial species.
• Achieved a combined measurement uncertainty of 10%-41% for quantification in bacterial extracts.

Conclusions:

• The developed method enables selective detection and quantification of bacterial cyclic dinucleotides and their isomers.
• The discovery of a novel cyclic di-AMP isomer highlights the complexity of bacterial signaling pathways.
• Normalization using total protein measurements provides a robust approach for comparing nucleotide concentrations across samples.