Diazobutanone-assisted isobaric labelling of phospholipids and sulfated glycolipids enables multiplexed quantitative lipidomics using tandem mass spectrometry.

Mass spectrometry-based quantitative lipidomics is an emerging field aiming to uncover the intricate relationships between lipidomes and disease development. However, quantifying lipidomes comprehensively in a high-throughput manner remains challenging owing to the diverse lipid structures. Here we propose a diazobutanone-assisted isobaric labelling strategy as a rapid and robust platform for multiplexed quantitative lipidomics across a broad range of lipid classes, including various phospholipids and glycolipids. The diazobutanone reagent is designed to conjugate with phosphodiester or sulfate groups, while accommodating various functional groups on different lipid classes, enabling subsequent isobaric labelling for high-throughput multiplexed quantitation. Our method demonstrates excellent performance in terms of labelling efficiency, detection sensitivity, quantitative accuracy and broad applicability to various biological samples. Finally, we performed a six-plex quantification analysis of lipid extracts from lean and obese mouse livers. In total, we identified and quantified 246 phospholipids in a high-throughput manner, revealing lipidomic changes that may be associated with obesity in mice.