Reference-pool discovery with cohort-wide signal extraction

What does this strategy enable?

This workflow separates discovery from cohort-wide quantification. Reference pools establish the target list; individual samples supply the quantitative measurements. Both annotated metabolites and retained unknown features can be measured. The method is not defined by the longevity cohort used to demonstrate it, and it does not require every feature to be identified.

Transferable workflow

Workflow

1
Prepare representative reference pools
Include relevant cohort subsets and acquire pooled discovery data, blanks and consistent QC samples.
2
Build the polar feature list
XCMS detects pooled features; CAMERA supports adduct selection. The study supplements these with AcquireX results.
xcms CAMERAAcquireX
3
Curate reference targets
Review artifacts and annotation evidence. The published polar branch uses DecoID; the lipid branch uses Lipid Annotator. Retain suitable unknown features too.
DecoIDLipid Annotator
4
Extract cohort peak areas
Use Skyline batch by batch with curated masses and retention-time boundaries; inspect integration and adjust for retention shifts.
Skyline
5
Evaluate correction and export measurements
Compare batch-correction choices against common QC samples before downstream biological analysis.
Published processing notebooks ↗

Connected resources

Tools used in this strategy

xcmsDetects features in pooled polar-metabolite data.View tool entry →CAMERASupports adduct selection during reference-list preparation.View tool entry →SkylineExtracts and reviews cohort-wide peak areas from the curated target list.View tool entry →AcquireXSupplies additional pooled-sample features in the published polar branch.DecoIDSupports spectral annotation of polar reference features.Lipid AnnotatorGenerates the published lipid reference list.Published processing notebooksReference processing, peak-boundary generation and batch-correction resources.External resource ↗

Strategy profile

Related objectives

Metabolite annotation

Components

Preprocessing and feature extraction, Quality control, Annotation or identification

Biological context

Broadly applicable

Ion mobility

Not specified

Scope and considerations

Designed for

Large LC-MS cohorts where pooled discovery can provide a reusable reference list.

Important requirements

Representative pools, blanks, shared QC material and individual-sample raw data.
Expert curation of reference peaks and integration boundaries.

Considerations

Rare or low-abundance compounds can be diluted below detection in pools and omitted from the target list.
Targeted signal extraction from untargeted data is not a validated absolute-concentration assay.
Check batch correction for preservation of biological differences; no normalization method is universally best.
The code is documented here, not executed or benchmarked by Navigator.

Reusable resources

✓ Code available✓ Published method available

Code↗Published method↗

Publication

Stancliffe E, Schwaiger-Haber M, et al. Analytical Chemistry 94, 17370-17378 (2022).

doi:10.1021/acs.analchem.2c01270