Shotgun Lipidomics Research Articles

Shotgun Lipidomics Combined with Laser Capture Microdissection: A Tool To Analyze Histological Zones in Cryosections of Tissues.

Shotgun analysis provides a quantitative snapshot of the lipidome composition of cells, tissues, or model organisms; however, it does not elucidate the spatial distribution of lipids. Here we demonstrate that shotgun analysis could quantify low-picomole amounts of lipids isolated by laser capture microdissection (LCM) of hundred micrometer-sized histological zones visualized at the cryosections of tissues. We identified metabolically distinct periportal (pp) and pericentral (pc) zones by immunostaining of 20 μm thick cryosections of a healthy mouse liver. LCM was used to ablate, catapult, and collect the tissue material from 10 to 20 individual zones covering a total area of 0.3-0.5 mm2 and containing ca. 500 cells. Top-down shotgun profiling relying upon computational stitching of 61 targeted selective ion monitoring ( t-SIM) spectra quantified more than 200 lipid species from 17 lipid classes including glycero- and glycerophospholipids, sphingolipids, cholesterol esters, and cholesterol. Shotgun LCM revealed the overall commonality of the full lipidome composition of pp and pc zones along with significant ( p < 0.001) difference in the relative abundance of 13 lipid species. Follow-up proteomics analyses of pellets recovered from an aqueous phase saved after the lipid extraction identified 13 known and 7 new protein markers exclusively present in pp or in pc zones and independently validated the specificity of their visualization, isolation, and histological assignment.

Dual Emitter Nano-Electrospray Ionization Coupled to Differential Ion Mobility Spectrometry-Mass Spectrometry for Shotgun Lipidomics.

Current lipidomics workflows are centered around acquisition of large data sets followed by lengthy data processing. A dual nESI-DIMS-MS platform was developed to perform real-time relative quantification between samples, providing data required for biomarker discovery and validation more quickly than traditional ESI-MS approaches. Nanosprayer activity and DIMS compensation field settings were controlled by a LabVIEW program synced to the accumulation portion of the ion trap scan function, allowing for full integration of the platform with a commercial mass spectrometer. By comparing samples with short electrospray pulses rather than constant electrospray, the DIMS and MS performance is normalized within an experiment, as signals are compared between individual mass spectra (ms time scale) rather than individual experiments (min-hr time scale). The platform was validated with lipid standards and extracts from nitrogen-deprived microalgae. Dual nESI-DIMS requires minimal system modification and is compatible with all traditional ion activation techniques and mass analyzers, making it a versatile improvement to shotgun lipidomics workflows.

Cold-Activated Lipid Dynamics in Adipose Tissue Highlights a Role for Cardiolipin in Thermogenic Metabolism

Thermogenic fat expends energy during cold for temperature homeostasis, and its activity regulates nutrient metabolism and insulin sensitivity. We measured cold-activated lipid landscapes in circulation and in adipose tissue by MS/MSALL shotgun lipidomics. We created an interactive online viewer to visualize the changes of specific lipid species in response to cold. In adipose tissue, among the approximately 1,600 lipid species profiled, we identified the biosynthetic pathway of the mitochondrial phospholipid cardiolipin as coordinately activated in brown and beige fat by cold in wild-type and transgenic mice with enhanced browning of white fat. Together, these data provide a comprehensive lipid bio-signature of thermogenic fat activation in circulation and tissue and suggest pathways regulated by cold exposure.

Two-Way Orthogonal Partial Least Squares (O2PLS) Analysis of the Lipidome and Transcriptome in Prediabetic and Diabetic Neuropathy

Peripheral neuropathy (PN) is a common complication of prediabetes and type 2 diabetes (T2D) for which there are limited treatment options. Using mouse models of prediabetes and T2D we have demonstrated that dietary reversal (DR) corrects the PN phenotype. To identify the mechanisms that underlie PN development and reversal, we employed an omics-based approach. This was achieved by integrating transcriptomic and lipidomic datasets derived from mouse sciatic nerve using two-way Orthogonal Partial Least Squares (O2PLS) analysis, a platform that is useful in uncovering the large-scale organization of metabolic networks. Sciatic nerve was isolated from five groups of mice that consisted of controls, high-fat (HF) fed mice (prediabetic), HF mice injected with low dose STZ (T2D), HF mice subjected to 8 week dietary reversal (DR) and T2D-DR. Tissue was processed for gene expression by mRNA-seq or for lipid species profiling by untargeted shotgun lipidomics. A total of 6,024 genes and 578 lipid species were selected and the systematic joint covariation between gene and lipid species were modeled using O2PLS. A clear separation occurred between T2D/prediabetes and healthy/DR mice. Compared to a traditional ANOVA analysis, we selected genes with the top 1% values (52 genes) as highly inter-correlated genes with lipid species; we additionally identified 28 more genes (53.8%) which also highly correlated with lipid species. The top 50 lipid species, including 29 novel species, were identified based on these analyses and primarily consisted of triacylglycerols (TG) and diacylglycerols (DG). Moreover, a comprehensive lipid-gene interaction network was constructed based on these genes and lipid species. This study demonstrates that O2PLS can provide significant and biologically relevant coordination between gene expression and lipid levels and can offer new insights into our understanding of the metabolic networks underlying PN in T2D. Disclosure K. Guo: None. E.L. Feldman: None. J. Hur: None.

Amelioration of Peripheral Neuropathy in Mouse Models of Diabetes by Dietary Reversal

Peripheral neuropathy (PN) is a common complication of prediabetes and type 2 diabetes (T2D). Lifestyle changes, including dietary reversal (DR), can reverse PN, although the mechanisms involved remain unclear. The goal of this study was to identify the mechanisms that underlie PN development in prediabetes and T2D and understand which of these are corrected upon DR. To achieve this, we used two models of PN and looked at the effects of DR on global gene expression and lipid profiles in peripheral nerves. C57BL/6J mice were fed a standard diet (10% kcal fat; SD) or high fat diet (60% kcal fat; HF) from 5 week. A subset of HF mice were injected with STZ at 12 week to induce hyperglycemia, reflecting a more diabetic-like phenotype. At 16 week, subsets of HF and HF-STZ mice were placed on the standard diet for 8 week (HF-DR and HF-STZ-DR, respectively) until 24 week when terminal phenotyping was performed. At baseline and at study completion, sciatic nerve tissue was processed for gene expression by RNAseq or for lipid species profiling by untargeted shotgun lipidomics. HF and HF-STZ mice display PN compared to controls. In contrast, peripheral nerve function of HF-DR and HF-STZ-DR mice was corrected upon DR. Neural gene expression analysis revealed numerous differentially expressed genes (DEG) when comparing SD to HF or HF-STZ mice. Interestingly, a proportion of the genes dysregulated in HF and HF-STZ, were similarly reversed by DR. KEGG enrichment of these genes revealed that lipid metabolism pathways were enriched. Lipidomic analysis revealed a similar pattern of differentially altered lipids that were reversed by DR in HF and HF-STZ mouse models. Lipids reversed by DR include diacylglycerol, a known mediator of insulin resistance. We demonstrate that DR of HF and HF-STZ mice can restore peripheral nerve function. We also show that genes related to lipid function are dysregulated in diseased states and are reversed by DR. This study supports the idea that DR improves peripheral nerve function by restoring lipid homeostasis. Disclosure P.D. O'Brien: None. K. Guo: None. L.M. Hinder: None. J.M. Hayes: None. F. Mendelson: None. M.A. Tabbey: None. J. Hur: None. E.L. Feldman: None.

Hepatic ketogenic insufficiency reprograms hepatic glycogen metabolism and the lipidome.

While several molecular targets are under consideration, mechanistic underpinnings of the transition from uncomplicated nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) remain unresolved. Here we apply multiscale chemical profiling technologies to mouse models of deranged hepatic ketogenesis to uncover potential NAFLD driver signatures. Use of stable-isotope tracers, quantitatively tracked by nuclear magnetic resonance (NMR) spectroscopy, supported previous observations that livers of wild-type mice maintained long term on a high-fat diet (HFD) exhibit a marked increase in hepatic energy charge. Fed-state ketogenesis rates increased nearly 3-fold in livers of HFD-fed mice, a greater proportionate increase than that observed for tricarboxylic acid (TCA) cycle flux, but both of these contributors to overall hepatic energy homeostasis fueled markedly increased hepatic glucose production (HGP). Thus, to selectively determine the role of the ketogenic conduit on HGP and oxidative hepatic fluxes, we studied a ketogenesis-insufficient mouse model generated by knockdown of the mitochondrial isoform of 3-hydroxymethylglutaryl-CoA synthase (HMGCS2). In response to ketogenic insufficiency, TCA cycle flux in the fed state doubled and HGP increased more than 60%, sourced by a 3-fold increase in glycogenolysis. Finally, high-resolution untargeted metabolomics and shotgun lipidomics performed using ketogenesis-insufficient livers in the fed state revealed accumulation of bis(monoacylglycero)phosphates, which also accumulated in livers of other models commonly used to study NAFLD. In summary, natural and interventional variations in ketogenesis in the fed state strongly influence hepatic energy homeostasis, glucose metabolism, and the lipidome. Importantly, HGP remains tightly linked to overall hepatic energy charge, which includes both terminal fat oxidation through the TCA cycle and partial oxidation via ketogenesis.

A phosphatidic acid-binding protein is important for lipid homeostasis and adaptation to anaerobic biofilm conditions in Pseudomonas aeruginosa.

A quantitative Pseudomonas aeruginosa proteomics approach revealed increased abundance of the so-far uncharacterized protein PA3911 in anaerobic biofilms grown under conditions of the cystic fibrosis lung. Physiological relevance of ORF PA3911 was demonstrated, inter alia, using phenotype microarray experiments. The mutant strain showed increased susceptibility in the presence of antimicrobials (minocycline, nafcillin, oxacillin, chloramphenicol and thiamphenicol), enhanced twitching motility and significantly impaired biofilm formation. PA3911 is a soluble, cytoplasmic protein in P. aeruginosa In protein-lipid overlay experiments, purified PA3911 bound specifically to phosphatidic acid (PA), the central hub of phospholipid metabolism. Structure-guided site-directed mutagenesis was used to explore the proposed ligand-binding cavity of PA3911. Protein variants of Leu56, Leu58, Val69 and Leu114 were shown to impair PA interaction. A comparative shotgun lipidomics approach demonstrated a multifaceted response of P. aeruginosa to anaerobic conditions at the lipid head group and fatty acid level. Lipid homeostasis in the PA3911 mutant strain was imbalanced with respect to lysophosphatidylcholine, phosphatidylcholine and diacylglycerol under anaerobic and/or aerobic conditions. The impact of the newly identified PA-binding protein on lipid homeostasis and the related macroscopic phenotypes of P. aeruginosa are discussed.

Sphingolipidomics of Thermotolerant Yeasts.

Mass spectrometry-based shotgun lipidomics was applied to the analysis of sphingolipids of 11 yeast strains belonging to four genera, that is Cryptococcus, Saccharomyces, Schizosaccharomyces, and Wickerhamomyces. The analysis yielded comprehensive results on both qualitative and quantitative representation of complex sphingolipids of three classes-phosphoinositol ceramide (PtdInsCer), mannosyl inositol phosphoceramide (MInsPCer), and mannosyl diinositol phosphoceramide (M(InsP)2 Cer). In total, nearly 150 molecular species of complex sphingolipids were identified. Individual strains were cultured at five different temperatures, that is 5, 10, 20, 30, and 40 °C (Wickerhamomyces genus only up to 30 °C), and the change in the culture temperature was found to affect the representation of both the sphingolipid classes and the length of the long-chain bases (LCB). Individual classes of sphingolipids differing in polar heads differed in the temperature response. The relative content of PtdInsCer increased with increasing temperature, whereas that of M(InsP)2 Cer decreased. Molecular species having C18-LCB were associated with low cultivation temperature, and a higher proportion of C20-LCB molecular species was produced at higher temperatures regardless of the type of polar head. On the other hand, the influence of temperature on the representation of very long-chain fatty acids (VLCFA) was less noticeable, the effect of the taxonomic affiliation of the strains being more pronounced than the cultivation temperature. For example, lignoceric and 2-hydrocylo-lignoceric acids were characteristic of the genera Cryptococcus and Schizosaccharomyces, and of Saccharomyces genus cultivated at high temperatures.

A novel sample preparation strategy for shotgun lipidomics of phospholipids employing multilamellar vesicles

The identification of lipids in biological samples is gaining importance. The advent of mass spectrometry-based lipidomics accelerated the field allowing nowadays for identification and quantification of complete lipidomes. However, due to solubility difficulties and varying properties of different lipid classes, sample preparation for lipidomics is still an issue. Of the many lipid classes, phospholipids are the major components of biological membranes. In solution, they spontaneously form lipid vesicles of various structures such as liposomes. They are therefore often used as membrane mimics when studying biological membranes and membrane proteins. Here, we present a novel sample preparation strategy for shotgun lipidomics employing liposomes prepared from lipid standards or lipid mixtures allowing the analysis of phospholipids directly from lipid bilayers. We validated our strategy for lipid identification by tandem mass spectrometry in positive or negative ion mode using different phospholipid species from various classes. We further tested our strategy for relative quantification by mixing different ratios of phospholipid species as well as determining the distribution of lipid species in a natural lipid extract.Graphical abstractᅟ

Characterization of lipid structures in femoral secretions of Galápagos marine iguanas by shotgun lipidomics

The lipidome of femoral gland secretions of the marine iguana (Amblyrhynchus cristatus) was studied. The main aim of this work was to identify and quantify the lipids present in femoral secretions using mass spectrometry-based shotgun lipidomics. Lipids belonging to seven lipid classes were identified: ceramides (43.41%), triacylglycerols (42.15%), cholesteryl esters (4.18%), sphingomyelins (3.61%), phosphatidylcholines (2.67%), phosphatidylinositols (2.50%) and phosphatidylethanolamines (1.48%). The second aim of the study was to explore whether lipid composition matches the different subspecies or populations of marine iguanas. The different subspecies of iguanas did not group together in relation to their lipidome. The authors’ findings are discussed in an ecological context and compared with the results of a previous study that characterized volatile lipids in marine iguana secretions using gas chromatography. Given the high diversity of lipid structures in femoral secretions discovered in this study, it is likely that some of these could be involved in intraspecific communication. Certain lipids could have other functions, such as building a barrier to avoid water loss. More data needs to be added to explore this hypothesis. This study is the first to use a lipidomics-based approach to characterize lipid composition in lizard femoral secretions. The application of shotgun lipidomics to reptilian glandular secretions will shed new light on the potential function of lipids in intraspecific chemical communication.

A Lipidomics Approach to Identifying Key Lipid Species Involved in VEGF-Inhibitor Mediated Attenuation of Bleomycin-Induced Pulmonary Fibrosis.

Poor molecular characterization of idiopathic pulmonary fibrosis (IPF) has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies and poor prognosis. Particularly, the role of lipid imbalance due to impaired lipid metabolism in the pathogenesis of IPF has been poorly studied. The authors have used shotgun lipidomics in a bleomycin (BLM) mouse model of pulmonary fibrosis with vascular endothelial growth factor (VEGF)-inhibitor CBO-P11 as a therapeutic measure, to identify a comprehensive set of lipids that contribute to the pathogenesis of pulmonary fibrosis. The authors report that attenuation of BLM-induced fibrotic response with CBO-P11 cotreatment is accompanied by a decrease in total lipid content and specific downregulation of lipids, which are upregulated in response to BLM treatment. Dysregulated lipids identified in this study hold the potential of being future biomarkers for IPF.

Adipose tissue ATGL modifies the cardiac lipidome in pressure-overload-induced left ventricular failure.

Adipose tissue lipolysis occurs during the development of heart failure as a consequence of chronic adrenergic stimulation. However, the impact of enhanced adipose triacylglycerol hydrolysis mediated by adipose triglyceride lipase (ATGL) on cardiac function is unclear. To investigate the role of adipose tissue lipolysis during heart failure, we generated mice with tissue-specific deletion of ATGL (atATGL-KO). atATGL-KO mice were subjected to transverse aortic constriction (TAC) to induce pressure-mediated cardiac failure. The cardiac mouse lipidome and the human plasma lipidome from healthy controls (n = 10) and patients with systolic heart failure (HFrEF, n = 13) were analyzed by MS-based shotgun lipidomics. TAC-induced increases in left ventricular mass (LVM) and diastolic LV inner diameter were significantly attenuated in atATGL-KO mice compared to wild type (wt) -mice. More importantly, atATGL-KO mice were protected against TAC-induced systolic LV failure. Perturbation of lipolysis in the adipose tissue of atATGL-KO mice resulted in the prevention of the major cardiac lipidome changes observed after TAC in wt-mice. Profound changes occurred in the lipid class of phosphatidylethanolamines (PE) in which multiple PE-species were markedly induced in failing wt-hearts, which was attenuated in atATGL-KO hearts. Moreover, selected heart failure-induced PE species in mouse hearts were also induced in plasma samples from patients with chronic heart failure. TAC-induced cardiac PE induction resulted in decreased PC/ PE-species ratios associated with increased apoptotic marker expression in failing wt-hearts, a process absent in atATGL-KO hearts. Perturbation of adipose tissue lipolysis by ATGL-deficiency ameliorated pressure-induced heart failure and the potentially deleterious cardiac lipidome changes that accompany this pathological process, namely the induction of specific PE species. Non-cardiac ATGL-mediated modulation of the cardiac lipidome may play an important role in the pathogenesis of chronic heart failure.

Adapted MS/MSALL Shotgun Lipidomics Approach for Analysis of Cardiolipin Molecular Species.

Cardiolipin (Ptd2 Gro) is a complex, doubly charged phospholipid located in the inner mitochondrial membrane where it plays an essential role in regulating bioenergetics. Abnormalities in Ptd2 Gro content or composition have been associated with mitochondrial dysfunction in a variety of disease states. Here, we report the development of an adapted high-resolution data-independent acquisition (DIA) MS/MSALL shotgun lipidomic method to enhance the accuracy and reproducibility of Ptd2 Gro molecular species quantitation from biological samples. Utilizing the doubly charged molecular ions and the isotopic pattern with negative mode electrospray ionization mass spectrometry (ESI-MS) using an adapted MS/MSALL approach, we profiled more than 150 individual Ptd2 Gro species, including monolysocardiolipin (MLPtd2 Gro). The method described in this study demonstrated high reproducibility, sensitivity, and throughput with a wide dynamic range. This high-resolution MS/MSALL shotgun lipidomics approach could be extended to screening aberrations of Ptd2 Gro metabolism involved in mitochondrial dysfunction in various pathological conditions and diseases.

Lipidomics Profiling of Myelin.

Lipidomics is a powerful approach that can provide quantitative characterization of hundreds of lipid species from biological samples. Recent studies have highlighted the value of lipidomics to study myelin biology. This chapter provides a detailed description for the application of multidimensional mass spectrometry shotgun lipidomics (MDMS-SL) to neuroscience research and particularly to the analysis of brain lipidomes with a particular emphasis on myelin lipids, from sample preparation to bioinformatics analyses. Sample preparation includes brain sample harvesting, homogenization, and lipid extraction. Lipid content is determined and quantified, in an unbiased manner and with wide coverage, using MDMS-SL. Overall, the approach described herein is applicable for whole brain tissue or specific brain regions (e.g., hippocampus, cerebellum), and is expected to yield new insights on various aspects of myelin biology and lipid metabolism.

Lipidomics reveals a systemic energy deficient state that precedes neurotoxicity in neonatal monkeys after sevoflurane exposure

Although numerous studies have raised public concerns regarding the safety of anesthetics including sevoflurane in children, the biochemical mechanisms leading to anesthetics-induced neurotoxicity remain elusive. Moreover, potential biomarker(s) for early detection of general anesthetics-induced brain injury are urgent for public health. We employed an enabling technology of shotgun lipidomics and analyzed nearly 20 classes and subclasses of lipids present in the blood serum of postnatal day (PND) 5 or 6 rhesus monkeys temporally collected after exposure to sevoflurane at a clinically relevant concentration or room-air as control. Lipidomics analysis revealed numerous significant anesthetic-induced changes of serum lipids and their metabolites as well as short chain acylcarnitines in the brain and cerebrospinal fluid after anesthetic exposure. These include decreased carnitine and acylcarnitines, unchanged triacylglycerol mass but accumulation of 16:0 and 18:1 fatty acyl chains in the triacylglycerol pool, losses of polyunsaturated fatty acids in both non-esterified fatty acid and phospholipid pools, and increased 4-hydroxynonenal content as early as 2 h after sevoflurane exposure. Importantly, the amounts of short chain acylcarnitines in the brain and cerebrospinal fluid were also significantly reduced after anesthetic exposure. We propose that this serum lipidomic profile can serve as indicative of neuronal damage. Our results reveal that sevoflurane exposure induces an energy deficient state in the brain evidenced by reduced free and acyl carnitine contents, as well as the presence of a pro-inflammatory state in the exposed animals, providing deep insights into the underlying mechanisms responsible for anesthetic-induced neurotoxicity.

Advances in chemical derivatization-based targeted lipidomics by mass spectrometric analysis

Lipidomics plays a significant role in the related studies of some serious diseases such as cardiovascular disease, diabetes and tumor, that has become a hot area of life science. Mass spectrometry is currently one of the core methods for lipidomics study. The strategy of chemical derivatization can remarkably improve the performance of mass spectrometric technologies for both shotgun and liquid chromatography-mass spectrometry-based lipidomics, and some interesting results have been reported. Therefore, this review will focus on the research progress in chemical derivatization-based targeted lipidomics by shotgun method and liquid chromatography-mass spectrometry, and the current problems as well as prospects in this area are also discussed. It should be helpful for the design and synthesis of new derivatization reagents, especially the stable isotope labeling derivatization reagents. The development and applications of new chemical derivatization strategies will provide useful techniques for lipidomics research.

Erratum to: Mechanism of Phospholipid Hydrolysis for Oyster Crassostrea plicatula Phospholipids During Storage Using Shotgun Lipidomics.

Erratum to: Lipids https://doi.org/10.1007/s11745-017-4305-7.

Effects of high-fat diet and age on the blood lipidome and circulating endocannabinoids of female C57BL/6 mice

Alterations in lipid metabolism play a significant role in the pathogenesis of obesity-associated disorders, and dysregulation of the lipidome across multiple diseases has prompted research to identify novel lipids indicative of disease progression. To address the significant gap in knowledge regarding the effect of age and diet on the blood lipidome, we used shotgun lipidomics with electrospray ionization-mass spectrometry (ESI-MS). We analyzed blood lipid profiles of female C57BL/6 mice following high-fat diet (HFD) and low-fat diet (LFD) consumption for short (6weeks), long (22weeks), and prolonged (36weeks) periods. We examined endocannabinoid levels, plasma esterase activity, liver homeostasis, and indices of glucose tolerance and insulin sensitivity to compare lipid alterations with metabolic dysregulation. Multivariate analysis indicated differences in dietary blood lipid profiles with the most notable differences after 6weeks along with robust alterations due to age. HFD altered phospholipids, fatty acyls, and glycerolipids. Endocannabinoid levels were affected in an age-dependent manner, while HFD increased plasma esterase activity at all time points, with the most pronounced effect at 6weeks. HFD-consumption also altered liver mRNA levels of PPARα, PPARγ, and CD36. These findings indicate an interaction between dietary fat consumption and aging with widespread effects on the lipidome, which may provide a basis for identification of female-specific obesity- and age-related lipid biomarkers.

Mechanism of Phospholipid Hydrolysis for Oyster Crassostrea plicatula Phospholipids During Storage Using Shotgun Lipidomics.

A fast and efficient shotgun lipidomics strategy was applied to analyze phospholipids (PL) in the oyster Crassostrea plicatula, including 29 species of phosphatidylcholine (PtdCho), 23 species of phosphatidylethanolamine (PtdEtn), 11 species of phosphatidylserine (PtdSer), 6 species of phosphatidylinositol (PtdIns), and 17 species of lysophospholipids (Lyso-PL). During storage at 4°C for 7days, the PL content decreased by 68.08%, but a significant increase in the FFA content was observed (from 63.11 to 318.72μg/g). PtdCho and PtdIns decreased relatively by 64.97 and 67.49%, and PtdSer decreased most markedly by 74.15%. However, the PtdEtn content increased slightly during the early stages of storage but subsequently began to decrease. Moreover, PL with eicosapentaenoic acid (EPA-PL) and docosahexaenoic acid (DHA-PL) decreased by 51.77 and 50.61%, whereas plasmalogens were relatively stable showing only a 25.46% decrease. In particular, through enzyme activity analysis of lipase, phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD), it was observed that the activities of all these enzymes increased at the early stage at 4°C, but their activities were at lower levels when the oysters were stored at -20°C. During the storage period at 4°C, correlation analysis suggests that the degradation of PtdCho was mostly correlated to PLA2 (p<0.05), whereas PtdEtn and PtdSer were more markedly correlated to lipase and PLD, respectively. The above result indicates that the hydrolysis mechanism of PL during seafood storage was correlated to the lipid hydrolytic enzyme activities under different storage temperatures.

Identification and Quantitation of C═C Location Isomers of Unsaturated Fatty Acids by Epoxidation Reaction and Tandem Mass Spectrometry.

Unsaturated fatty acids (FAs) serve as nutrients, energy sources, and signaling molecules for organisms, which are the major components for a large variety of lipids. However, structural characterization and quantitation of unsaturated FAs by mass spectrometry remain an analytical challenge. Here, we report the coupling of epoxidation reaction of the C═C in unsaturated FAs and tandem mass spectrometry (MS) for rapid and accurate identification and quantitation of C═C isomers of FAs in a shotgun lipidomics approach. Epoxidation of the C═C leads to the production of an epoxide which, upon collision induced dissociation (CID), produces abundant diagnostic ions indicative of the C═C location. The total intensity of the same set of diagnostic ions for one specific FA C═C isomer was also used for its relative and absolute quantitation. The simple experimental setup, rapid reaction kinetics (<2 min), high reaction yield (>90% for monounsaturated FAs), and easy-to-interpret tandem MS spectra enable a promising methodology particularly for the analysis of unsaturated FAs in complex biological samples such as human plasma and animal tissues.