Tracer-assisted shotgun lipidomics (TASL): A quantitative workflow integrating stable-isotope tracing with global...

Shotgun lipidomics provides a quantitative, steady-state overview of global lipidomes, but offers limited insight into metabolic dynamics. Tracer lipidomics yields time-resolved quantitative information on specific biosynthetic pathways, but labeling can perturb lipidomes, making labeled time-course samples unsuitable for steady-state comparisons. Here, we introduce Tracer-Assisted Shotgun Lipidomics (TASL), a strategy that integrates stable-isotope tracing with shotgun lipidomics in a single workflow, enabling time-resolved analysis while retaining labeled samples as inputs for steady-state lipidome profiling. This is achieved through a minimally perturbing strategy where cells are pre-equilibrated in an unlabeled precursor before switching to the isotopically labeled precursor at the same concentration.

As a proof of concept, TASL was applied to HCT116 colorectal cancer cells and three drug-resistant variants, sampled over 24 h following the switch from unlabeled l-serine to l-serine-(¹³C₃¹⁵N) to label de novo synthesized sphingolipids. Leveraging the enhanced statistical power of this design, global steady-state analysis revealed accumulation of dihydrosphingolipid species lacking the canonical 4,5-trans double bond in their long-chain base as the most prominent alteration shared across drug-resistant cell lines. Time-resolved analysis of the de novo sphingolipid biosynthesis pathway subsequently identified a pronounced bottleneck at dihydroceramide desaturation, diverting flux toward dihydrosphingomyelin despite an otherwise intact pathway.

Together, TASL provides a generalizable and minimally perturbing framework for integrating global steady-state lipidomics with time-resolved pathway analysis, and can be readily extended to other tracers, pathways, and biological systems to study metabolic rewiring at the lipidome scale.