Click chemistry-based enrichment strategy for tracing cellular fatty acid metabolism by LC-MS/MS
Rujie Yang, Jian Zou, Jia-Yue Liu, Jiangkun Dai, Jian‐Bo Wan
Abstract
Fatty acids (FAs), which were initially recognized as energy sources and essential building blocks of biomembranes, serve as the precursors of important signaling molecules. Tracing FA metabolism is essential to understanding the biochemical activity and role of FAs in physiological and pathological events. Inspired by the advances in click chemistry for protein enrichment, we herein established a click chemistry-based enrichment (CCBE) strategy for tracing the cellular metabolism of eicosapentaenoic acid (EPA, 20:5 n-3) in neural cells. Terminal alkyne-labeled EPA (EPAA) used as a surrogate was incubated with N2a, mouse neuroblastoma cells, and alkyne-labeled metabolites (ALMs) were selectively captured by an azide-modified resin via a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction for enrichment. After removing unlabeled metabolites, ALMs containing a triazole moiety were cleaved from solid-phase resins and subjected to liquid chromatograph mass spectrometry (LC-MS) analysis. The proposed CCBE strategy is highly selective for capturing and enriching alkyne-labeled metabolites from complicated matrices. In addition, the method can overcome current detection limits by improving analytical performance, including MS sensitivity of targets, and chromatographic separation of sn-position glycerophospholipid regioisomers, facilitating structural characterization of ALM by a specific MS/MS fragmentation signature, and versatile fluorescence detection of ALM for cellular distribution. This CCBE strategy might be expanded to trace the metabolism of other FAs, small molecules, or drugs.