Herein, we report the development of an online-coupled LC–PB–MS/MS platform and corresponding analytical workflows, aiming to achieve large-scale lipid analysis at C=C location level in a sensitive and high-throughput fashion. Considering that liquid chromatography (LC–)MS is one of the most commonly used analytical platforms for large-scale lipidomic studies, merging the PB reaction onto the LC–MS/MS platform would provide a key solution to unsaturated lipid analysis at C=C location level. The capabilities of PB–MS/MS for localization of C=C in unsaturated lipids (free fatty acids (FAs), cholesterol esters, and glycerol phospholipids (GPs)) 21– 24 and quantitation of lipid C=C location isomers have been demonstrated using the shotgun lipid analysis approach, where crude lipid extract is directly subjected for MS analysis without a prior separation. In earlier studies, we have developed a strategy that combines solution C=C specific derivatization (the Paternò–Büchi (PB) reaction) with conventional MS/MS methods via low-energy CID, termed as PB–MS/MS. While the newly developed gas-phase fragmentation methods are promising in terms of indeendent identification of unknown structures from complex mixtures, their capability in global identification of unsaturated lipids remains to be demonstrated. These include C=C specific chemical derivatization or cleavage prior to MS 12– 14, coupling high resolving power ion mobility separation with MS 15, and alternative gas-phase ion activation methods either producing fragments specific to a C=C 16– 19 or inducing extensive fragmentation along the acyl chain 20.
Several MS strategies have attempted to address this challenge. Considering that the biophysical properties of unsaturated lipids are closely modulated by the number, locations, and configurations of C=Cs in acyl/alkyl chains 11, research in lipidomics is hampered by the limited knowledge about the exact structures of unsaturated lipids. This limitation stems from employing low-energy collision-induced dissociation (CID) as the tandem mass spectrometry (MS/MS) technique in conventional lipidomics analysis workflows, which fails in providing the information for locating C=C bonds. With the development of modern mass spectrometry and data analysis tools, large-scale lipid profiling can be routinely practiced at fatty acyl/alkyl compositional level 1, 10, but not at the C=C location level. Lipid profiles generated by MS are increasingly utilized in metabolic flux analysis, tissue imaging, and disease biomarker discovery 8, 9.Įven though lipids, using glycerophospholipids as an example, are assembled from simple chemical building blocks, a plethora of structural possibilities exist when considering different combinations of acyl/alkyl chains, their linkage positions on the glycerol backbone ( sn-position), and the occurrence of carbon–carbon double bonds (C=C) and stereo-centers. Mass spectrometry (MS) has become the enabling tool for lipidomics due to the capability of identifying and quantifying lipids in complex mixtures at high sensitivity and throughput 1. Therefore, mapping the complete molecular composition of a lipidome is considered as an important goal of lipidomics in order to further understand pathways and mechanisms behind lipid homeostasis 7.
On the other hand, disturbed lipid homeostasis is often linked to the occurrence and development of a pathological state, as commonly found for cancer 4, insulin-resistant diabetes 5, and cardiovascular diseases 6. The exact lipid composition, however, is closely regulated for maintaining appropriate cell functions regardless of differences in feed nutrients 3. The cellular lipidome is highly complex, characterized by the presence of thousands of molecular species with concentrations spanning three to five orders of magnitude 2. Lipids are an essential class of biomolecules that are heavily recruited in cell structuring, signaling, organelle compartmentalization, and energy production in different biological systems 1.
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