Due to the diversity of lipid structures, comprehensive high‑throughput quantification of the lipidome remains challenging. Recently, the research group of Li Lingjun at the University of Wisconsin–Madison published an article in *Nature Chemistry* titled “Diazobutanone‑assisted isobaric labelling of phospholipids and sulfated glycolipids enables multiplexed quantitative lipidomics using tandem mass spectrometry”. By combining diazobutanone with aminoxy tandem mass tags (aminoxyTMT) for high‑throughput quantification, the authors established a platform for distinguishing multiple lipid classes and achieved multiplexed quantitative lipidomics analysis. This method features high labelling efficiency, high detection sensitivity, accurate quantification, and good biological compatibility. In this study, the authors performed six‑plex quantitative analysis on liver lipid extracts from lean and obese mice, identified and quantified 246 phospholipids in high throughput, and revealed lipidomic changes potentially associated with obesity in mice.

Isobaric tagging allows quantitative comparison of multiple samples in a single experiment; different mass tags provide higher quantitative precision, reproducibility, and sample throughput. Phospholipids (PLs) and glycolipids are major components of the lipidome, regulating membrane dynamics, serving as energy reservoirs, and acting as precursors of bioactive metabolites. For phospholipids, structural diversity poses challenges for targeting different PL classes. Due to the poor nucleophilicity of phospholipids, O‑alkylation of their common phosphodiester groups for subsequent labelling is difficult. The authors designed a diazo compound, diazobutanone, which enables a diazo‑assisted isobaric tagging strategy for multiplexed quantification of all phosphate‑ and sulfate‑containing lipids.

Diazobutanone is a volatile compound synthesized in only two steps. It enables O‑alkylation of PLs and specific reaction with aminoxyTMT, while its volatility simplifies purification by evaporation. In principle, phospholipids first react with diazobutanone to introduce carbonyl groups, which then react with the mass tag aminoxyTMT to form oxime linkages as labels. The highly specific and efficient oxime formation preserves other functional groups on phospholipids and sulfated glycolipids. To demonstrate broad applicability, nine representative phosphate‑containing lipid classes were tested, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), lysophosphatidylcholine (LysoPC), ether‑phosphatidylcholine (etherPC), and sphingomyelin (SM). Excess reagent can be removed by evaporation, reducing sample loss and compatibility with downstream mass spectrometry. Before labelling, phospholipids and sulfated glycolipids are typically detected as negative ions; after phosphate derivatization and aminoxyTMT labelling with tertiary amines, tagged lipids are readily analyzed in positive mode.

Reaction conditions including temperature, solvent, and catalyst loading were optimized to achieve high conversion and easy vacuum purification for quantitative analysis. Notably, diazobutanone selectively targets phosphate and sulfate groups under optimized conditions, leaving other functional groups intact, such as esters, hydroxyls, amines, amides, carboxylates, alkenes, and quaternary amines. This avoids complex side products that reduce analytical sensitivity.

Labelled lipids were characterized with a mass increment of 372 Da from butanone and aminoxyTMT; PA showed a 744 Da increment due to double tagging, with minor single‑tagged PA. Previous methods using diazomethane often produced isobaric interferences between modified PS, PC, and PA, requiring offline fractionation. In this work, no amine alkylation was observed and PL fragmentation was minimal, likely due to lower reactivity of diazobutanone and milder conditions.

Finally, six‑plex quantitative lipidomics was applied to liver lipids from lean and obese mice. Deuterated internal standards were added before homogenization for recovery correction. After Folch extraction, samples were tagged with diazobutanone and aminoxyTMT, pooled, and analyzed by LC‑MS/MS. PLs were identified by accurate mass and diagnostic fragments, and reporter ion intensities were extracted across six channels. A total of 251 acyl‑chain‑defined PL species were identified. In obese mice, 79 PL classes and 146 PL molecular species were enriched, whereas 18 classes and 28 species were enriched in lean mice. The PC/PE ratio, a liver disease indicator, slightly decreased in obese mice, consistent with insulin resistance without severe hepatic pathology. Hierarchical clustering showed good intra‑group reproducibility and clear separation between groups, revealing four major lipid clusters: one lean‑enriched, two obese‑enriched, and one unchanged. PLs with long acyl chains (>38 carbons) increased in obesity, while short‑chain PLs (<34 carbons) and highly unsaturated PC decreased, a pattern also seen in hepatic disease, suggesting that acyl chain elongation may affect liver function in obesity.

In summary, the diazobutanone‑assisted isobaric tagging strategy enables multiplexed quantitative lipidomics of phospholipids and glycolipids.