Isotope Tracing
For stable-isotope labeling experiments (¹³C, ¹⁵N, ²H, ¹⁸O, ³⁴S), LEAF can extract and quantify isotopologue distributions — the M+0, M+1, M+2, ... peaks — alongside the parent compound.
[Screenshot: Tracing Editor on the Extract page with default 13C offsets]
Set up tracing
The Tracing Editor panel sits on the right side of the Extract page, below the parameters sidebar. By default it's pre-configured for ¹³C tracing (M+1 and M+2):
| Isotopologue | Mass offset (Da) | Description |
|---|---|---|
| M+1 | 1.003355 | One ¹³C incorporation |
| M+2 | 2.006710 | Two ¹³C incorporations |
To add more isotopologues, click Add Tracer and adjust the isotope counts.
Supported isotopes
| Isotope | Symbol | Mass shift per atom |
|---|---|---|
| Carbon-13 | ¹³C | 1.003355 Da |
| Nitrogen-15 | ¹⁵N | 0.997035 Da |
| Oxygen-18 | ¹⁸O | 2.004244 Da |
| Sulfur-34 | ³⁴S | 1.995796 Da |
| Deuterium | ²H | 1.006277 Da |
You can mix isotopes within a single experiment — e.g., a ¹³C₅¹⁵N glutamate tracer needs both isotopes counted.
Common configurations
Standard ¹³C-glucose tracing
The default M+1 and M+2 are usually enough.
Fully labeled substrates (e.g., U-¹³C₆-glucose)
Add tracers up to M+6 for glucose, or however many carbons your substrate has.
Multiple tracers in one experiment
Add a separate tracer entry per labeling pattern. The Tracing Editor handles them independently.
Save and reuse tracing configs
| Button | Effect |
|---|---|
| Export | Save the current tracing setup as a JSON file |
| Import | Load a saved JSON config |
| Reset to default | Revert to the M+1 / M+2 ¹³C defaults |
Sharing the JSON file with a collaborator means they can run the exact same isotope setup without re-entering anything.
Read isotopologue distributions
After extraction, the Isotopologue Bar Chart (bottom-left panel of the Peak Picking view) shows the distribution per metabolite.
[Screenshot: isotopologue bar chart in percentage mode with grouped samples]
| Color | Isotopologue |
|---|---|
| Blue | M+0 (unlabeled parent) |
| Green | M+1 |
| Amber | M+2 |
| Rose | M+3 |
| Purple | M+4+ |
Absolute vs percentage
- Absolute intensity — raw peak heights. Useful for comparing total pool sizes.
- Percentage — fractional labeling (what % of the metabolite pool carries each isotopologue). Useful for comparing labeling between conditions.
When sample grouping is on, bars show mean ± SEM per group — the standard way to present tracing data.
Tips
- Validate your CSV first — formula errors break isotope mass calculations
- Use 5 ppm mass tolerance or tighter — heavier isotopologues are close in mass and a wide window picks up noise
- Watch for natural abundance — even unlabeled samples show small M+1 peaks (~1.1% per carbon). LEAF does not currently correct for natural abundance; you'll need to do this downstream if your science demands it.
Export tracing data
In the export dialog, ensure Include isotopologues is on. The CSV gets one row per (compound × isotopologue × sample). The .msd always includes them when present. See Export.
Next step
→ Untargeted feature discovery — for hypothesis-free analyses