What does this strategy enable?
This strategy adapts native electrospray concepts to metabolomics so that protonated and metal-bound forms of the same chromatographic analyte can be detected together. Post-column pH adjustment and infusion of a selected metal are coupled to feature processing and ion identity molecular networking, allowing candidate metal-binding molecules to be prioritized from characteristic mass offsets and shared chromatographic behavior.
Transferable workflow
Workflow
- 1
Separate metabolites by LC
Analyze the extract using an LC-MS method suitable for retaining native-like metal-binding interactions after chromatographic separation.
- 2
Adjust post-column pH
Infuse a volatile base or buffer after the LC column to establish conditions compatible with observing metal complexes during electrospray.
- 3
Infuse the metal of interest
Introduce a metal salt post-column so protonated and metal-bound forms of candidate ligands can be observed with matched retention behavior.
- 4
Process features and ion identities
Detect and align chromatographic features and group coeluting ion species with correlated peak shapes and expected mass relationships.
- 5
Build an ion identity molecular network
Use ion identity molecular networking to connect ion species and spectral relationships, highlighting candidate metal-binding compounds.
- 6
Prioritize and validate candidate binders
Interpret expected metal-adduct mass offsets, inspect spectra and chromatograms, and validate prioritized compounds with standards or orthogonal evidence where available.
Connected resources
Tools used in this strategy
Strategy profile
Related objectives
Native metabolomics, Natural-product discovery
Components
Chromatography, Post-column modification or infusion, Data acquisition, Preprocessing and feature extraction, Annotation or identification, Molecular networking, Data and workflow sharing
Sample types
Microbial culture or biomass, Water or aquatic sample, Synthetic or analytical standards
Biological context
Microbial, Fungal, Marine, Environmental, Synthetic or reference material
Ion mobility
Ion mobility not required
Scope and considerations
Designed for
- Discovering metal-binding small molecules in complex extracts using standard LC-MS hardware with post-column modification.
- Comparing protonated and metal-bound ion species through chromatographic correlation and defined mass offsets.
Important requirements
- LC-MS system with a post-column setup that permits pH adjustment and metal-salt infusion.
- Appropriate blanks and controls to distinguish biologically relevant complexes from background or source-derived adducts.
Considerations
- Metal identity, pH, salt concentration and electrospray conditions can alter complex formation and should be optimized and reported.
- The network highlights candidate binding behavior; structural identity and biological relevance require additional evidence.
Reusable resources
Publication
Aron AT, Petras D, Schmid R, et al. Native mass spectrometry-based metabolomics identifies metal-binding compounds. Nature Chemistry 14, 100–109 (2022).