Volume 7, Issue 6 2400039
RESEARCH ARTICLE

Profiling of cyclic di-adenyl and -guanyl nucleotides and their precursors and degradation products in bacteria using LC–MS/MS

Silvio Uhlig

Corresponding Author

Silvio Uhlig

Nordic Institute of Dental Materials, Oslo, Norway

Correspondence

Silvio Uhlig, Nordic Institute of Dental Materials, Sognsveien 70A, 0855 Oslo, Norway.

Email: [email protected]

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Kun Cai

Kun Cai

Institute of Oral Biology, University of Oslo, Oslo, Norway

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Krystyna Anna Liskiewicz

Krystyna Anna Liskiewicz

Institute of Oral Biology, University of Oslo, Oslo, Norway

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Maria Pain

Maria Pain

Nordic Institute of Dental Materials, Oslo, Norway

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Lene Aiko Grutle

Lene Aiko Grutle

Nordic Institute of Dental Materials, Oslo, Norway

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Hanne Røberg-Larsen

Hanne Røberg-Larsen

Section for Chemical Life Sciences—Biomolecules, Bio-Inspired Materials and Bioanalytics, University of Oslo, Oslo, Norway

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Roger Simm

Roger Simm

Institute of Oral Biology, University of Oslo, Oslo, Norway

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First published: 22 February 2024
Citations: 2

Abstract

The 3,3′-linked cyclic dinucleotides (CDNs) of adenosine monophosphate (AMP) and guanosine monophosphate (GMP), cyclic di-AMP (c-di-AMP), cyclic di-GMP (c-di-GMP), and c-GMP–AMP (cGAMP) are second messenger molecules in bacteria that regulate processes, such as biofilm formation, motility, virulence, stress response, and cell wall homeostasis. To analyze the profiles of the three CDNs together with their breakdown and precursor molecules, 5′-phosphoadenylyl-(3′ → 5′)adenine (pApA), 5′-guanylyl-(3′ → 5′)guanine (pGpG), 5′-AMP, 3′- and 5′-GMP, adenosine triphosphate (ATP), and GTP, we established an LC–MS/MS-based approach for semi-quantification and profiling. Weak anion exchange solid-phase extraction was employed to improve selectivity and instrumental signal/noise of CDNs as well as pApA and pGpG. CDNs were analyzed using reverse-phase UHPLC–MS/MS, whereas all other nucleotides were analyzed using hydrophilic interaction chromatography (HILIC)–MS/MS. The instrument limit of quantification ranged from 0.72 (c-di-AMP) to 60 nM (ATP and GTP). We applied this method to the analysis of the nine nucleotides in eight bacterial strains and found that the profiles varied widely in terms of both absolute and relative concentrations. Thus, CDN concentrations were generally <1 pmol/mg biomass, and the hydrolysis products, pApA and pGpG, were detected at lower pmol/mg concentrations. The presented method is a relatively simple and straightforward approach to profiling nucleotides with the rationale of comparing their relative levels between populations of bacterial strains.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.