High-throughput relative quantification of fatty acids by 12-plex isobaric labeling and microchip capillary electrophoresis - Mass spectrometry

Fatty acids (FAs) are essential cellular components and play important roles in various biological processes. Importantly, FAs produced by microorganisms from renewable sugars are considered sustainable substrates for biodiesels and oleochemicals. Their complex structures and diverse functional roles in biochemical processes necessitate the development of efficient and accurate methods for their quantitative analysis. Here, we developed a novel method for relative quantification of FAs by combining 12-plex isobaric N,N-dimethyl leucine-derivatized ethylenediamine (DiLeuEN) labeling and microchip capillary electrophoresis-mass spectrometry (CE-MS). This method enables simultaneous quantification of 12 samples in a single MS analysis. DiLeuEN labeling introduced tertiary amine center structure into FAs, which makes them compatible with the positive mode separation of commercial microchip CE systems and further improves the sensitivity. The CE separation parameters were optimized, and the quantification accuracy was assessed using FA standards. Microchip CE-MS detection exhibited high sensitivity with a femtomole level detection limit and a total analysis time within 8 min. Finally, the applicability of our method to complex biological samples was demonstrated by analyzing FAs produced by four industrially relevant yeast strains (Saccharomyces cerevisiae, Yarrowia lipolytica YB-432, Yarrowia lipolytica Po1f and Rhodotorula glutinis). The analysis time for each sample is less than 1 min. This work addresses the current challenges in the field by introducing a method that combines microchip-based capillary electrophoresis separation with multiplex isobaric labeling. Our method not only offers remarkable sensitivity and rapid analysis speed but also the capability to quantify fatty acids across multiple samples simultaneously, which holds significant potential for extensive application in FA quantitative studies in diverse research areas, promising an enhanced understanding of FA functions and mechanisms. [Display omitted] •Integrated 12-plex DiLeuEN isobaric labeling with CE-MS to enable rapid and precise fatty acids (FAs) relative quantification.•Enhanced the throughput of FAs quantification with notable precision and accuracy through 12-plex DiLeuEN isobaric labeling.•Achieved fast FAs separation in 8 min with fmol level detection limits via microchip CE-MS.•Successfully applied the developed method for quantitative analysis of FAs across four different yeast