What does this strategy enable?
Stable-isotope labeling–assisted molecular networking layers biosynthetic incorporation information onto MS/MS similarity. A labeled precursor such as a stable-isotope amino acid is supplied to a producing organism, labeled mass shifts are detected in metabolite features, and molecular networking groups related MS/MS spectra. The combination helps distinguish biosynthetically connected analogues and can provide sequence or building-block information that molecular networking alone does not supply.
Transferable workflow
Workflow
- 1
Select and feed a stable-isotope precursor
Add a labeled biosynthetic building block to the culture under conditions that permit incorporation into specialized metabolites, while retaining appropriate unlabeled controls.
- 2
Detect isotope incorporation
Compare labeled and unlabeled LC-MS data to identify features with mass shifts consistent with incorporation of the supplied precursor.
- 3
Acquire and compare MS/MS spectra
Collect fragmentation spectra for candidate labeled and related metabolites.
- 4
Build a molecular network
Organize structurally related spectra by MS/MS similarity and overlay isotope-incorporation evidence to recognize families sharing the labeled building block.
- 5
Infer biosynthetic or structural relationships
Use the combined label pattern and fragmentation relationships to prioritize analogues, infer building-block composition, and guide targeted structural validation.
Connected resources
Tools used in this strategy
Strategy profile
Related objectives
Natural-product discovery, Molecular-family discovery, Structure elucidation
Components
Sample preparation, Chromatography, Data acquisition, Preprocessing and feature extraction, Molecular networking, Annotation or identification
Sample types
Microbial culture or biomass
Biological context
Fungal, Microbial
Organisms
Aspergillus nidulans
Ion mobility
Ion mobility not required
Scope and considerations
Designed for
- Linking natural-product analogues to a supplied biosynthetic precursor.
- Characterizing families of peptide or other metabolites where isotope incorporation provides interpretable biosynthetic information.
Important requirements
- A biologically plausible labeled precursor and culture system capable of incorporating it.
- Matched labeled and unlabeled controls and sufficient mass accuracy to resolve the expected isotope shift.
Considerations
- Label incorporation may be incomplete, diluted by metabolism, or redistributed into downstream metabolites.
- Molecular similarity and label incorporation support a biosynthetic relationship but do not by themselves establish complete pathway genetics or final structure.
Reusable resources
Publication
Klitgaard A, Nielsen JB, Frandsen RJN, Andersen MR, Nielsen KF. Combining Stable Isotope Labeling and Molecular Networking for Biosynthetic Pathway Characterization. Analytical Chemistry 87, 6520–6526 (2015).