Isotope Tracing

For stable-isotope labeling experiments (¹³C, ¹⁵N, ²H, ¹⁸O, ³⁴S), LEAF extracts and quantifies isotopologue distributions (M+0, M+1, M+2, ...) 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. By default, it is 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 additional isotopologues, click Add Tracer and set the isotope count.

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

Multiple isotope types can be represented in one experiment. For example, a ¹³C₅¹⁵N glutamate tracer requires both carbon and nitrogen counts.

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 to the carbon count of the labeled substrate.

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

The exported JSON file records the isotope setup and can be reused in later runs.

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, suitable for comparing total pool size.
• Percentage — fractional labeling, suitable for comparing isotope incorporation between conditions.

When sample grouping is enabled, bars show mean ± SEM per group.

Natural-abundance correction

LEAF can apply natural-abundance correction to targeted tracing results after extraction. Correction is configured per analysis and stored inside the .msd archive as a correction_config.json sidecar.

In the web UI:

1. Open the isotopologue bar chart gear popover, or the export panel.
2. Click Configure tracers….
3. Add each tracer element and purity, for example 13C at 0.99.
4. Enable correction separately for the bar plot preview or for CSV export.

The bar plot falls back to raw values with an inline note if correction is enabled before a tracer is configured. Corrected CSV export requires a tracer config.

Current correction support:

Supported	Notes
Tracer elements	C, H, N
Mode	High-resolution correction
Labeling model	Uniform labeling for all atoms of the configured tracer element

Not covered by the current correction surface: O / S multi-heavy-isotope correction, low-resolution correction, and position-specific labeling.

Tips

• Validate the CSV before extraction — formula errors invalidate isotope mass calculations
• Use 5 ppm mass tolerance or tighter — heavier isotopologues are close in mass and a wide window may include unrelated signal
• Account for natural abundance — unlabeled samples exhibit M+1 contributions of approximately 1.1% per carbon from natural ¹³C. Use LEAF's correction toggle for supported C / H / N tracer experiments; use a specialized downstream tool when the experimental design falls outside that support.

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.

Also from a script

The Tracing Editor exports a JSON config that the CLI accepts directly:

leaf targeted ./samples ./compounds.csv ./outputs \
  --polarity NEG --tracing-path ./tracing-13C.json

For corrected CSV output:

leaf targeted ./samples ./compounds.csv ./outputs \
  --polarity NEG --tracing-path ./tracing-13C.json \
  --correct --tracer 13C:0.99

→ leaf targeted reference Or in Python: Recipe 4 — Tracing in a script

Next step

→ Export targeted results