Identification and quantitation of lipid C=C location isomers: A shotgun lipidomics approach enabled by photochemical reaction.
ABSTRACT: The field of lipidomics has been significantly advanced by mass spectrometric analysis. The distinction and quantitation of the unsaturated lipid isomers, however, remain a long-standing challenge. In this study, we have developed an analytical tool for both identification and quantitation of lipid C=C location isomers from complex mixtures using online Paternò-Büchi reaction coupled with tandem mass spectrometry (MS/MS). The potential of this method has been demonstrated with an implementation into shotgun lipid analysis of animal tissues. Among 96 of the unsaturated fatty acids and glycerophospholipids identified from rat brain tissue, 50% of them were found as mixtures of C=C location isomers; for the first time, to our knowledge, the quantitative information of lipid C=C isomers from a broad range of classes was obtained. This method also enabled facile cross-tissue examinations, which revealed significant changes in C=C location isomer compositions of a series of fatty acids and glycerophospholipid (GP) species between the normal and cancerous tissues.
Project description:Lipid dysregulation has been implicated in multiple sclerosis due to its involvement during and after inflammation. In this study, we have profiled fatty acids (FAs) in the mouse model of multiple sclerosis with new capabilities of assigning carbon-carbon double bond (C=C) location(s) and quantifying C=C location isomers. These new capabilities are enabled by pairing the solution phase Paternò-Büchi (PB) reaction that modifies C=C bonds in FAs, with tandem mass spectrometry (MS/MS), termed as PB-MS/MS. A series of unsaturated FAs and C=C location isomers have been identified, including FA17:1 (?10), FA18:1 (?9 and ?11), FA18:2 (?9 and ?12), and FA 20:4 (?5, ?8, ?11, ?14). Notable differences in saturated and unsaturated FAs between normal and experimental autoimmune encephalomyelitis (EAE) mice spinal cords have been detected. Furthermore, the effects of hydralazine, a scavenger of acrolein, on profile changes of FAs in mice were studied. Increased ?11-to-?9 isomer ratios for FA 18:1 were noted in the diseased samples as compared to the control. The present work provides a facile and robust analytical method for the quantitation of unsaturated FAs as well as identification of FA C=C location isomers, which will facilitate discovering prospective lipid markers in multiple sclerosis.
Project description:Lipid desaturation plays important roles in biological processes and the disease states. Here, we report a simple but efficient method for mapping unsaturated phospholipids including the spatial distribution of lipid C=C location isomers in animal organs by coupling the C=C specific derivatization with direct analysis mass spectrometry (MS). Lipids are sampled directly by a stainless-steel wire from rat brain or kidney, extracted, and derivatized via the Paternò-Büchi reaction in a glass emitter of the nanoelectrospray ionization (nanoESI) source. Subsequent analysis by nanoESI-tandem mass spectrometry reveals C=C locations and relative quantities of lipid C=C location isomers. Unsaturated lipids, such as phospholipids and free fatty acids, have been identified with ion intensities spanning two orders of magnitude in rat brain. Typical sample consumption is less than 10 ?g/measurement and the time for each analysis is about 3 min. This method should serve as a complementary method to high spatial resolution mass spectrometry imaging techniques, because it offers a streamlined experimental workflow for rapid profiling of lipids with C=C specificity to enable such applications as point-of-care disease diagnostics.
Project description:Mass spectrometry-based lipidomics is the primary tool for the structural analysis of lipids but the effective localization of carbon-carbon double bonds (C=C) in unsaturated lipids to distinguish C=C location isomers remains challenging. Here, we develop a large-scale lipid analysis platform by coupling online C=C derivatization through the Paternò-Büchi reaction with liquid chromatography-tandem mass spectrometry. This provides rich information on lipid C=C location isomers, revealing C=C locations for more than 200 unsaturated glycerophospholipids in bovine liver among which we identify 55 groups of C=C location isomers. By analyzing tissue samples of patients with breast cancer and type 2 diabetes plasma samples, we find that the ratios of C=C isomers are much less affected by interpersonal variations than their individual abundances, suggesting that isomer ratios may be used for the discovery of lipid biomarkers.
Project description:Mass spectrometry analysis of cholesteryl esters (CEs) faces several challenges, with one of them being the determination of the carbon-carbon double bond (C=C) locations within unsaturated fatty acyl chains. Patern?-Büchi (PB) reaction, a photochemical reaction based on the addition of acetone to C=C, is capable of C=C location determination when coupled with tandem mass spectrometry (MS/MS). In this study, the PB reaction conditions were tailored for CEs and subsequent nanoelectrospray ionization (nanoESI). A solvent system containing acetone/methanol/dichloromethane/water (40/30/20/10, volume ratios) and 100 ?M LiOH was determined to be optimal, resulting in reasonable PB reaction yield (~30%) and good ionization efficiency (forming lithium adduct of CEs). Collision-induced dissociation (CID) of the PB reaction products produced characteristic fragment ions of CE together with those modified by the PB reactions, such as lithiated fatty acyl ([FA + Li]+) and its PB product ([FA - PB + Li]+). MS3 CID of [FA - PB + Li]+ led to abundant C=C diagnostic ion formation, which was used for C=C location determination and isomer quantitation. A PB-MS3 CID approach was developed and applied for CE analysis from human plasma. A series of unsaturated CEs was identified with specific C=C locations within fatty acyl chains. Absolute quantitation for each CE species was achieved including coexisting C=C location isomers, such as ?9 and ?11 isomers of CE 18:1 and ?-6 and ?-3 isomers of CE 18:3. These results show that PB-MS/MS is useful in uncovering structural diversity of CEs due to unsaturation in fatty acyls, which is often undetected from current lipid analysis approach. Graphical Abstract ?.
Project description:A porous polymer coating transfer enrichment method is developed for the direct mass spectrometry (MS) analysis of lipids. The enrichment is fast (ca. 1?min) and enables the profiling and quantitation of lipids in small-volume biofluid samples. Coupled with a photochemical Paternò-Büchi reaction, this method enables the fast determination of lipid structure at the C=C location level and point-of-care lipid biomarker analysis.
Project description:Shotgun lipid analysis based on electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is increasingly used in lipidomic studies. One challenge for the shotgun approach is the discrimination of lipid isomers and isobars. Gas-phase charge inversion via ion/ion reactions has been used as an effective method to identify multiple isomeric/isobaric components in a single MS peak by exploiting the distinctive functionality of different lipid classes. In doing so, fatty acyl chain information can be obtained without recourse to condensed-phase separations or derivatization. This method alone, however, cannot provide carbon-carbon double bond (C=C) location information from fatty acyl chains. Herein, we provide an enhanced method pairing photochemical derivatization of C=C via the Paternò-Büchi reaction with charge inversion ion/ion tandem mass spectrometry. This method was able to provide gas-phase separation of phosphatidylcholines and phosphatidylethanolamines, the fatty acyl compositions, and the C=C location within each fatty acyl chain. We have successfully applied this method to bovine liver lipid extracts and identified 40 molecular species of glycerophospholipids with detailed structural information including head group, fatty acyl composition, and C=C location. Graphical Abstract ?.
Project description:As a major class of mammalian lipids, phosphatidylcholines (PCs) often contain mixtures of structural isomers, resulting from different lipogenesis pathways. Profiling PCs at the isomer level, however, remains challenging in lipidomic settings, especially for characterizing the positions of fatty acyls on the glycerol backbone (sn-positions) and the locations of carbon-carbon double bonds (C[double bond, length as m-dash]Cs) in unsaturated acyl chains. In this work, we have developed a workflow for profiling PCs down to sn- and C[double bond, length as m-dash]C locations at high coverage and sensitivity. This capability is enabled by radical-directed fragmentation, forming sn-1 specific fragment ions upon collision-induced dissociation (CID) of bicarbonate anion adducts of PCs ([M + HCO3]-) inside a mass spectrometer. This new tandem mass spectrometry (MS/MS) method can be simply incorporated into liquid chromatography by employing ammonium bicarbonate in the mobile phase without any instrument modification needed. It is also compatible with the online Paternò-Büchì reaction and subsequent MS/MS for the assignment of C[double bond, length as m-dash]C locations in sn-1 fatty acyl chains of unsaturated PCs. The analytical performance of the workflow is manifested by identification of 82 distinct PC molecular species from the polar extract of bovine liver, including quantification of 19 pairs of sn-isomers. Finally, we demonstrate that five pairs of PC sn-isomers show significant compositional changes in tissue samples of human breast cancer relative to controls, suggesting a potential for monitoring PC sn-isomers for biomedical applications.
Project description:Lipids play a pivotal role in biological processes and lipid analysis by mass spectrometry (MS) has significantly advanced lipidomic studies. While the structure specificity of lipid analysis proves to be critical for studying the biological functions of lipids, current mainstream methods for large-scale lipid analysis can only identify the lipid classes and fatty acyl chains, leaving the C=C location and sn-position unidentified. In this study, combining photochemistry and tandem MS we develop a simple but effective workflow to enable large-scale and near-complete lipid structure characterization with a powerful capability of identifying C=C location(s) and sn-position(s) simultaneously. Quantitation of lipid structure isomers at multiple levels of specificity is achieved and different subtypes of human breast cancer cells are successfully discriminated. Remarkably, human lung cancer tissues can only be distinguished from adjacent normal tissues using quantitative results of both lipid C=C location and sn-position isomers.
Project description:Fatty acid (FA) profiling provides phenotypic information and is increasingly used in a broad range of biological and biomedical studies. Quantitation of unsaturated FAs with confident carbon-carbon double bond (C?C) location assignment is both sample and time consuming using traditional gas chromatography mass spectrometry analysis. In this study, we developed a rapid, sensitive, and quantitative method for profiling unsaturated FAs without using chromatographic separations. This method was based on a combination of in-solution photochemical tagging of a C?C in FAs and a subsequent gas-phase detagging via tandem (neutral loss scan) mass spectrometry. It enabled quantitation of unsaturated FAs from various biological samples (blood, plasma, and cell lines). More importantly, quantitative information on FA C?C location isomers, which was traditionally overlooked, could now be obtained and applied to studying FA changes between normal and cancerous human prostate cells.
Project description:Single cell MS (SCMS) techniques are under rapid development for molecular analysis of individual cells among heterogeneous populations. Lipids are basic cellular constituents playing essential functions in energy storage and the cellular signaling processes of cells. Unsaturated lipids are characterized with one or multiple carbon-carbon double (C?C) bonds, and they are critical for cell functions and human diseases. Characterizing unsaturated lipids in single cells allows for better understanding of metabolomic biomarkers and therapeutic targets of rare cells (e.g., cancer stem cells); however, these studies remain challenging. We developed a new technique using a micropipette needle, in which Paternò-Büchi (PB) reactions at C?C bond can be induced, to determine locations of C?C bonds in unsaturated lipids at the single-cell level. The micropipette needle is produced by combining a pulled glass capillary needle with a fused silica capillary. Cell lysis solvent and PB reagent (acetone or benzophenone) are delivered into the micropipette needle (tip size ? 15 um) through a fused silica capillary. The capillary needle plays multiple functions (i.e., single cell sampling probe, cell lysis container, microreactor, and nano-ESI emitter) in the experiments. Both regular (no reaction) and reactive (with PB reaction) SCMS analyses of the same cell can be achieved. C?C bond locations were determined from MS scan and MS/MS of PB products assisted by Python programs. This technique can potentially be used for other reactive SCMS studies to enhance molecular analysis for broad ranges of single cells.