Isobaric Intramolecular Paterno–Buchi Reactions Enable Multiplexed Quantitation of Fatty Acids down to C═C Locations

Abstract Fatty acids (FAs), as key lipids in energy storage and signaling, are dynamically regulated to maintain cellular homeostasis. FA profiling has become a valuable approach for investigating lipid reprogramming in disease. Although isomer-resolved mass spectrometry (MS) methods facilitate differentiation of FA isomers, quantitation is limited by the scarcity of isomer standards. Here, leveraging isobaric intramolecular Paternò–Büchi (i2PB) reactions, we develop an isomer-resolved multiplexed quantitation method for FA profiling. The i2PB reagents, synthesized via amidation of benzoyl-phenylalanine with the 6-plex TMT tags at near-quantitative conversion, overcome the low-yield limitations of conventional PB reactions for low-abundance lipids. Rapid i2PB reactions (4 s) enable online integration with liquid chromatography, while subsequent MS2 and MS3 analyses generate reporter ions for 6-channel FA quantitation at the sum composition and C═C location levels, respectively. For FA quantitation, this approach offers improved throughput, amol sensitivity, and a linear dynamic range of 2 orders of magnitude. Using i2PB, we achieve parallel quantitation of over 100 FAs with defined C═C locations in normal and breast cancer cells, providing new insights into metabolic reprogramming of FAs in breast cancer cells.