Abstract
Gangliosides are vital cell membrane components whose metabolic dysregulation is implicated in various cancers. However, a systems-level understanding of their metabolism has been hindered by their structural complexity and low cellular abundance. Herein, we have developed a deep profiling workflow for gangliosides that integrates selective enrichment, liquid chromatography-ion mobility spectrometry, and isomer-resolved tandem mass spectrometry. This workflow enhances detection sensitivity 100-fold, enabling the identification of 391 ganglioside structures in a human breast adenocarcinoma cell line (MCF-7) at multiple structural levels. We further reveal coordinated remodeling of gangliosides in MCF-7 cancer cells, including shifts toward a-series glycans, increased incorporation of long-chain sphingosine bases, and altered C[double bond, length as m-dash]C location isomers. By integrating these lipidomic findings with targeted gene expression analysis and quantitative proteomics, we reconstruct a ganglioside biosynthetic network that delineates dysregulation across five key structural modules. This lipid-centric approach offers new insights into the metabolic reprogramming of gangliosides and holds potential for studying lipid metabolism in diverse diseases.