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Omics analyses collectively refer to the possibility of profiling genetic variants, RNA, epigenetic markers, proteins, lipids, and metabolites. The most common analytical approaches used for detecting molecules present within biofluids related to metabolism are vibrational spectroscopy techniques, represented by infrared, Raman, and nuclear magnetic resonance (NMR) spectroscopies and mass spectrometry (MS). Omics-based assessments utilizing MS are rapidly expanding and being applied to various scientific disciplines and clinical settings. Most of the omics instruments are operated by specialists in dedicated laboratories; however, the development of miniature portable omics has made the technology more available to users for field applications. Variations in molecular information gained from omics approaches are useful for evaluating human health following environmental exposure and the development and progression of numerous diseases. As MS technology develops so do statistical and machine learning methods for the detection of molecular deviations from personalized metabolism, which are correlated to altered health conditions, and they are intended to provide a multi-disciplinary overview for researchers interested in adding multiomic analysis to their current efforts. This includes an introduction to mass spectrometry-based omics technologies, current state-of-the-art capabilities and their respective strengths and limitations for surveying molecular information. Furthermore, we describe how knowledge gained from these assessments can be applied to personalized medicine and diagnostic strategies.
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Exposición a Riesgos Ambientales , Espectrometría de Masas , Metabolómica , Humanos , Espectrometría de Masas/métodos , Exposición a Riesgos Ambientales/análisis , Metabolómica/métodos , Proteómica/métodos , Biomarcadores , Genómica/métodosRESUMEN
Lysosomes are degradative organelles that facilitate the removal and recycling of potentially cytotoxic materials and mediate a variety of other cellular processes, such as nutrient sensing, intracellular signaling, and lipid metabolism. Due to these central roles, lysosome dysfunction can lead to deleterious outcomes, including the accumulation of cytotoxic material, inflammation, and cell death. We previously reported that cationic amphiphilic drugs, such as imipramine, alter pH and lipid metabolism within macrophage lysosomes. Therefore, the ability for imipramine to induce changes to the lipid content of isolated macrophage lysosomes was investigated, focusing on sphingomyelin, cholesterol, and glycerophospholipid metabolism as these lipid classes have important roles in inflammation and disease. The lysosomes were isolated from control and imipramine-treated macrophages using density gradient ultracentrifugation, and mass spectrometry was used to measure the changes in their lipid composition. An unsupervised hierarchical cluster analysis revealed a clear differentiation between the imipramine-treated and control lysosomes. There was a significant overall increase in the abundance of specific lipids mostly composed of cholesterol esters, sphingomyelins, and phosphatidylcholines, while lysophosphatidylcholines and ceramides were overall decreased. These results support the conclusion that imipramine's ability to change the lysosomal pH inhibits multiple pH-sensitive enzymes in macrophage lysosomes.
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Imipramina , Esfingomielinas , Humanos , Esfingomielinas/metabolismo , Imipramina/farmacología , Colesterol/metabolismo , Macrófagos/metabolismo , Lisosomas/metabolismo , Inflamación/metabolismo , Metabolismo de los Lípidos , Glicerofosfolípidos/metabolismoRESUMEN
Multiple reaction monitoring (MRM) profiling is a strategy for the exploratory analysis of small molecules and lipids by direct sample injection, ie, without the use of chromatographic separation. It is based on instrument methods that comprise a list of ion transitions (MRMs), in which the precursor ion is the expected ionized m/z of the lipid at its species level, ie, the description of lipid class and number of carbon and double bonds in the fatty acid chain(s), and the product ion is a fragment expected for the lipid class or for the fatty acid neutral loss. The Lipid Maps database is expanding constantly, and therefore the MRM-profiling methods associated with this database need to be continuously updated. Here, we provide a comprehensive overview and the key references for the MRM-profiling methodology and workflow, followed by a step-by-step approach to build MRM-profiling instrument acquisition methods for class-based lipid exploratory analysis based on the Lipid Maps database. The detailed workflow includes (1) importing the list of lipids from the database; (2) for a given class, combining isomeric lipids described at full structural level into 1 entry to obtain the neutral mass at species level; (3) attributing the standard Lipid Maps abbreviated nomenclature for the lipid at its species level; (4) predicting the ionized precursor ions; and (5) adding the expected product ion. We also describe how to simulate the precursor ion for the suspect screening of modified lipids using lipid oxidation and their expected product ions as an example. After determining the MRMs, information about collision energy, dwell time, and other instrument parameters are added to finalize the acquisition method. As an example of final method output, we describe the format for Agilent MassHunter v.B.06 and provide the parameters in which optimization can be performed by lipid class using one or more lipid standards.
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Carbono , Ácidos Grasos , Espectrometría de Masas , Bases de Datos Factuales , IsomerismoRESUMEN
Lipid homeostasis is critical for maintaining normal cellular functions including membrane structural integrity, cell metabolism, and signal transduction. Adipose tissue and skeletal muscle are two major tissues involved in lipid metabolism. Adipose tissue can store excessive lipids in the form of triacylglyceride (TG), which can be hydrolyzed to release free fatty acids (FFAs) under insufficient nutrition states. In the highly energy-demanding skeletal muscle, lipids serve as oxidative substrates for energy production but can cause muscle dysfunction when overloaded. Lipids undergo fascinating cycles of biogenesis and degradation depending on physiological demands, while dysregulation of lipid metabolism has been increasingly recognized as a hallmark of diseases such as obesity and insulin resistance. Thus, it is important to understand the diversity and dynamics of lipid composition in adipose tissue and skeletal muscle. Here, we describe the use of multiple reaction monitoring profiling, based on lipid class and fatty acyl chain specific fragmentation, to explore various classes of lipids in skeletal muscle and adipose tissues. We provide a detailed method for exploratory analysis of acylcarnitine (AC), ceramide (Cer), cholesteryl ester (CE), diacylglyceride (DG), FFA, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), and TG. Characterization of lipid composition within adipose tissue and skeletal muscle under different physiological situations will provide biomarkers and therapeutic targets for obesity-related diseases.
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Lipidómica , Obesidad , Humanos , Obesidad/metabolismo , Ésteres del Colesterol , Metabolismo de los Lípidos , Fosfatidilcolinas/metabolismo , Músculo Esquelético/metabolismoRESUMEN
Our objective is to evaluate the effects of feeding rumen-protected Met (RPM) throughout the transition period and early lactation on the lipid profile of the preimplantation embryos and the endometrial tissue of Holstein cows. Treatments consisted of feeding a total mixed ration with top-dressed RPM (Smartamine® M, Adisseo, Alpharetta, GA, United States; MET; n = 11; RPM at a rate of 0.08% of DM: Lys:Met = 2.8:1) or not (CON; n = 9, Lys:Met = 3.5:1). Endometrial biopsies were performed at 15, 30, and 73 days in milk (DIM). Prior to the endometrial biopsy at 73 DIM, preimplantation embryos were harvested via flushing. Endometrial lipid profiles were analyzed using multiple reaction monitoring-profiling and lipid profiles of embryos were acquired using matrix assisted laser desorption/ionization mass spectrometry. Relative intensities levels were used for principal component analysis. Embryos from cows in MET had greater concentration of polyunsaturated lipids than embryos from cows in CON. The endometrial tissue samples from cows in MET had lesser concentrations of unsaturated and monounsaturated lipids at 15 DIM, and greater concentration of saturated, unsaturated (specifically diacylglycerol), and monounsaturated (primarily ceramides) lipids at 30 DIM than the endometrial tissue samples from cows in CON. In conclusion, feeding RPM during the transition period and early lactation altered specific lipid classes and lipid unsaturation level of preimplantation embryos and endometrial tissue.
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Fatty acids are an important source of energy and a key component of phospholipids in membranes and organelles. Saturated fatty acids (SFAs) are converted into unsaturated fatty acids (UFAs) by stearoyl Co-A desaturase (SCD), an enzyme active in cancer. Here, we studied how the dynamics between SFAs and UFAs regulated by SCD impacts ovarian cancer cell survival and tumor progression. SCD depletion or inhibition caused lower levels of UFAs vs. SFAs and altered fatty acyl chain plasticity, as demonstrated by lipidomics and stimulated Raman scattering (SRS) microscopy. Further, increased levels of SFAs resulting from SCD knockdown triggered endoplasmic reticulum (ER) stress response with brisk activation of IRE1α/XBP1 and PERK/eIF2α/ATF4 axes. Disorganized ER membrane was visualized by electron microscopy and SRS imaging in ovarian cancer cells in which SCD was knocked down. The induction of long-term mild ER stress or short-time severe ER stress by the increased levels of SFAs and loss of UFAs led to cell death. However, ER stress and apoptosis could be readily rescued by supplementation with UFAs and reequilibration of SFA/UFA levels. The effects of SCD knockdown or inhibition observed in vitro translated into suppression of intraperitoneal tumor growth in ovarian cancer xenograft models. Furthermore, a combined intervention using an SCD inhibitor and an SFA-enriched diet initiated ER stress in tumors growing in vivo and potently blocked their dissemination. In all, our data support SCD as a key regulator of the cancer cell fate under metabolic stress and point to treatment strategies targeting the lipid balance.
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Supervivencia Celular , Endorribonucleasas , Ácidos Grasos Insaturados , Neoplasias Ováricas , Progresión de la Enfermedad , Ácido Graso Desaturasas , Ácidos Grasos/farmacología , Ácidos Grasos Insaturados/farmacología , Femenino , Humanos , Fosfolípidos , Proteínas Serina-Treonina Quinasas , Estearoil-CoA Desaturasa/metabolismoRESUMEN
The aim of this work was to compare the lipidome and metabolome profiling in the Longissimus thoracis muscle early and late postmortem from high and normal ultimate pH (pHu) beef. Lipid profiling discriminated between high and normal pHu beef based on fatty acid metabolism and mitochondrial beta-oxidation of long chain saturated fatty acids at 30 min postmortem, and phospholipid biosynthesis at 44 h postmortem. Metabolite profiling also discriminated between high and normal pHu beef, mainly through glutathione, purine, arginine and proline, and glycine, serine and threonine metabolisms at 30 min postmortem, and glycolysis, TCA cycle, glutathione, tyrosine, and pyruvate metabolisms at 44 h postmortem. Lipid and metabolite profiles showed reduced glycolysis and increased use of alternative energy metabolic processes that were central to differentiating high and normal pHu beef. Phospholipid biosynthesis modification suggested high pHu beef experienced greater oxidative stress.
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Lipidómica , Metaboloma , Animales , Bovinos , Concentración de Iones de Hidrógeno , Glutatión/metabolismo , Fosfolípidos , Músculo Esquelético/metabolismoRESUMEN
Nanoparticles (NPs) interact with biomolecules by forming a biocorona (BC) on their surface after introduction into the body and alter cell interactions and toxicity. Metabolic syndrome (MetS) is a prevalent condition and enhances susceptibility to inhaled exposures. We hypothesize that distinct NP-biomolecule interactions occur in the lungs due to MetS resulting in the formation of unique NP-BCs contributing to enhanced toxicity. Bronchoalveolar lavage fluid (BALF) was collected from healthy and MetS mouse models and used to evaluate variations in the BC formation on 20 nm iron oxide (Fe3O4) NPs. Fe3O4 NPs without or with BCs were characterized for hydrodynamic size and zeta potential. Unique and differentially associated proteins and lipids with the Fe3O4 NPs were identified through proteomic and lipidomic analyses to evaluate BC alterations based on disease state. A mouse macrophage cell line was utilized to examine alterations in cell interactions and toxicity due to BCs. Exposures to 6.25, 12.5, 25, and 50 µg/mL of Fe3O4 NPs with BCs for 1 h or 24 h did not demonstrate overt cytotoxicity. Macrophages increasingly associated Fe3O4 NPs following addition of the MetS BC compared to the healthy BC. Macrophages exposed to Fe3O4 NPs with a MetS-BC for 1 h or 24 h at a concentration of 25 µg/mL demonstrated enhanced gene expression of inflammatory markers: CCL2, IL-6, and TNF-α compared to Fe3O4 NPs with a healthy BC. Western blot analysis revealed activation of STAT3, NF-κB, and ERK pathways due to the MetS-BC. Specifically, the Jak/Stat pathway was the most upregulated inflammatory pathway following exposure to NPs with a MetS BC. Overall, our study suggests the formation of distinct BCs due to NP exposure in MetS, which may contribute to exacerbated inflammatory effects and susceptibility.
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Thousands of chemical compounds produced by industry are dispersed in the human environment widely enough to reach the world population, and the introduction of new chemicals constantly occurs. As new synthetic molecules emerge, rapid analytical workflows for screening possible presence of exogenous compounds in biofluids can be useful as a first pass analysis to detect chemical exposure and guide the development and application of more elaborate LC-MS/MS methods for quantification. In this study, a suspect screening workflow using the multiple reaction monitoring (MRM) profiling method is proposed as a first pass exploratory technique to survey selected exogenous molecules in human urine samples. The workflow was applied to investigate 12 human urine samples using 310 MRMs related to the chemical functionalities of 87 exogenous compounds present in the METLIN database and reported in the literature. A total of 11 MRMs associated with five different compounds were detected in the samples. Product ion scans for the precursor ions of the selected MRMs were acquired as a further identification step for these chemicals. The suspect screening results suggested the presence of five exogenous compounds in the human urine samples analyzed, namely metformin, metoprolol, acetaminophen, paraxanthine and acrylamide. LC-MS/MS was applied as a last step to confirm these results, and the presence of four out of the five targets selected by MRM profiling were corroborated, indicating that this workflow can support the selection of suspect compounds to screen complex samples and guide more time-consuming and specific quantification analyses.
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Espectrometría de Masas en Tándem , Cromatografía Liquida/métodos , Bases de Datos Factuales , Humanos , Espectrometría de Masas en Tándem/métodos , Flujo de TrabajoRESUMEN
RESEARCH QUESTION: Can exposure time to equilibration solutions during oocyte vitrification affect the lipid profile of oocytes and embryonic development? Could vitrification media supplemented with oleic, linoleic acids and L-carnitine effectively minimize damage induced by vitrification on embryo development and oocyte membrane lipid profile? DESIGN: Experimental study including 936 oocytes from C57BL/6J mice, randomly divided into fresh IVF (control) and equilibration solution groups. Oocytes were exposed to equilibration solution from Irvine Scientific, Tvitri-4 or Tvitri-4 supplemented with L-carnitine and fatty acids for 7 or 10 min, vitrified-warmed, and submitted to IVF. The lipid profile of oocytes immediately after equilibration solution exposure was also asessed using the same equilibration times and solution compositions. RESULTS: Longer equilibration time resulted in lower oocyte survival and blastocyst rates, and reduced relative abundance of structural lipids, i.e. phosphatidylcholines and sphingomyelins, varying according to equilibration solution composition. It also induced membrane disruptions resembling bubbles in the oocyte surface predominantly in equilibration solution from Irvine Scientific, rarely in Tvitri-4 and absent in Tvitri-4 supplemented with L-carnitine and fatty acids. To reveal the metabolic pathways associated with the equilibration phase of vitrification, lipid pathway analysis was conducted; both P-values and pathway impact values showed that the linoleic acid metabolism (Pâ¯=â¯0.00223; impact =1) and alpha-Linolenic acid metabolism (Pâ¯=â¯0.00084; impactâ¯=â¯0.33) were the most pathway perturbed, followed by glycerophospholipid metabolism (Pâ¯=â¯0.0167; impactâ¯=â¯0.25) CONCLUSION: A longer equilibration phase pre-vitrification can influence embryo development and induce changes in oocyte lipid composition related to membrane integrity. The results suggest internalization of oleic and linoleic acids added to equilibration solution by the oocyte, which, to some extent, contributed to membrane phospholipids preservation, regardless of the equilibration times assessed.
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Criopreservación , Vitrificación , Animales , Carnitina/farmacología , Criopreservación/métodos , Desarrollo Embrionario , Ácidos Grasos/farmacología , Femenino , Humanos , Ácidos Linoleicos/farmacología , Ratones , Ratones Endogámicos C57BL , Oocitos , EmbarazoRESUMEN
The embryonic environment can modify cancer cell metabolism, and it is reported to induce the loss of tumorigenic properties and even affect the differentiation of cancer cells into normal tissues. The cellular mechanisms related to this remarkable phenomenon, which is likely mediated by cell-to-cell communication, have been previously investigated with particular focus on the proteins and genes involved. In this study we report the optimization and results of a straightforward in vitro system where mouse prostate carcinoma (RM-1) cells were co-cultured for three days with preimplantation mouse embryos or spiked with deproteinated extracts from mouse blastocysts. Compared to controls, both treatments induced RM-1 cells to increase the expression of the SOX-2 gene, which is related to cellular stemness, as well as altered their lipid composition. Specific acyl-carnitines, diacylglycerols, phosphatidylglycerols, phosphatidylinositols, phosphatidylserines and cardiolipins selected using an elastic net model discriminated the treated RM-1 cells from controls. Note that the tumorigenic properties of the treated RM-1 cells were not evaluated in this research. Due to the nature of the lipids impacted in the treated RM-1 cells, we hypothesize that mitochondrial metabolism has been altered, and that small molecules both secreted from and present within the embryos might be involved in the induction of metabolic changes observed in the RM-1 cells. These molecules, which could influence cancer cell metabolism, may still be unknown (i.e. structure, role).
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Blastocisto , Desarrollo Embrionario , Animales , Blastocisto/metabolismo , Técnicas de Cocultivo , Desarrollo Embrionario/genética , Lípidos , Masculino , RatonesRESUMEN
PURPOSE: We developed an accessible method for labeling small extracellular vesicles (sEVs) without disrupting endogenous ligands. Using labeled sEVs administered to conscious rats, we developed a multiple compartment pharmacokinetic model to identify potential differences in the disposition of sEVs from three different cell types. METHODS: Crude sEVs were labeled with a non-homologous oligonucleotide and isolated from cell culture media using a commercial reagent. Jugular vein catheters were used to introduce EVs to conscious rats (n = 30) and to collect blood samples. Digital PCR was leveraged to allow for quantification over a wide dynamic range. Non-linear mixed effects analysis with first order conditional estimation - extended least squares (FOCE ELS) was used to estimate population-level parameters with associated intra-animal variability. RESULTS: 86.5% ± 1.5% (mean ± S.E.) of EV particles were in the 45-195 nm size range and demonstrated protein and lipid markers of endosomal origin. Incorporated oligonucleotide was stable in blood and detectable over five half-lives. Data were best described by a three-compartment model with one elimination from the central compartment. We performed an observation-based simulated posterior predictive evaluation with prediction-corrected visual predictive check. Covariate and bootstrap analyses identified cell type having an influence on peripheral volumes (V2 and V3) and clearance (Cl3). CONCLUSIONS: Our method relies upon established laboratory techniques, can be tailored to a variety of biological questions regarding the pharmacokinetic disposition of extracellular vesicles, and will provide a complementary approach for the of study EV ligand-receptor interactions in the context of EV uptake and targeted therapeutics.
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Vesículas Extracelulares/metabolismo , Nanopartículas/metabolismo , Oligonucleótidos/farmacocinética , Animales , Secuencia de Bases , Transporte Biológico , Caenorhabditis elegans/genética , Humanos , Ligandos , Lípidos/química , Masculino , MicroARNs , Modelos Biológicos , Oligonucleótidos/metabolismo , Ratas Sprague-Dawley , Imagen Individual de MoléculaRESUMEN
In this study, we applied multiple reaction monitoring (MRM)-profiling to explore the relative ion intensity of lipid classes in plasma samples from sea turtles in order to profile lipids relevant to sea turtle physiology and investigate how dynamic ocean environments affect these profiles. We collected plasma samples from foraging green (Chelonia mydas, n = 28) and hawksbill (Eretmochelys imbricata, n = 16) turtles live captured in North Pacific Costa Rica in 2017. From these samples, we identified 623 MRMs belonging to 10 lipid classes (sphingomyelin, phosphatidylcholine, free fatty acid, cholesteryl ester, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, ceramide, and triacylglyceride) and one metabolite group (acyl-carnitine) present in sea turtle plasma. The relative ion intensities of most lipids (80%) were consistent between species, across seasons, and were not correlated to body size or estimated sex. Of the differences we observed, the most pronounced was the differences in relative ion intensity between species. We identified 123 lipids that had species-specific relative ion intensities. While some of this variability is likely due to green and hawksbill turtles consuming different food items, we found indications of a phylogenetic component as well. Of these, we identified 47 lipids that varied by season, most belonging to the structural phospholipid classes. Overall, more lipids (n = 39) had higher relative ion intensity in the upwelling (colder) season compared to the non-upwelling season (n = 8). Further, we found more variability in hawksbill turtles than green turtles. Here, we provide the framework in which to apply future lipid profiling in the assessment of health, physiology, and behavior in endangered sea turtles.
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Lípidos/sangre , Filogenia , Especificidad de la Especie , Tortugas/genética , Animales , Clima , Costa Rica , Lípidos/clasificación , Lípidos/genética , Estaciones del Año , Tortugas/fisiologíaRESUMEN
A marker indicative of the fertility potential of replacement gilts early in development would decrease culling rates in the sow herd, improve sow herd reproductive efficiency, and reduce production costs. The objective of this study was to determine if vaginal lipid profiles at 21 d postnatal (PN) could predict sow reproductive performance. Vaginal swabs of the anterior vagina were taken at 21 ± 4 d PN from gilts born on a commercial sow production facility for lipidomic analysis. Animals were followed prospectively for 2 yr and assigned to reproductive performance categories based on the observation of estrus or piglets weaned per sow per year (PSY) across two farrowings. Lipids were extracted from cellular material collected with swabs taken from high fertility (HF; n = 28; ≥26 PSY) and infertile (IF; n = 34; no estrus, no pregnancy) animals, and multiple reaction monitoring profiling was used for lipidome analysis. The relative abundance of arachidonic acid (C20:4) and docosahexaenoic acid (C22:6) was lower (P < 0.05) in IF gilts than HF gilts, whereas the abundance of the free fatty acids such as cerotic (C26:0), ximenic (C26:1), and nonadecanoic (C19:0) acids was greater (P < 0.05) in IF gilts. Additionally, eicosapentaenoic acid (C20:5), a precursor of prostaglandins, was higher (P < 0.05) in IF gilts. The perspective of having a panel of lipids captured with vaginal swabs at weaning that can predict the reproductive efficiency of gilts shows promise and warrants future research in this area.
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Lipidómica , Reproducción , Animales , Biomarcadores , Femenino , Fertilidad , Tamaño de la Camada , Paridad , Embarazo , Porcinos , Vagina , DesteteRESUMEN
Small molecules, including metabolites and lipids, provide information on metabolic pathways and active biological processes in living organisms. They are often diagnostic of disease. Current exploratory methods for metabolomics and lipidomics mostly rely on separation using liquid or gas chromatography (LC or GC) coupled with mass spectrometers capable of acquiring high resolution data to generate an enormous data, but at the cost of lengthy processing and data acquisition. Even though many molecules can be identified and quantified by these methods, the laborious protocols for purification, identification, and validation limit the accessible sample chemical information. To improve the speed and efficiency of exploratory metabolomics and lipidomics, multiple reaction monitoring profiling (MRM profiling) has been developed. This strategy involves a three-stage workflow which starts by considering the metabolome as a collection of functional groups. The Discovery Stage interrogates a representative sample mixture for functional groups using the functional group specific precursor ion (Prec) scans and neutral loss (NL) scans. This experiment usually uses a triple quadrupole mass spectrometer without chromatography, i.e. by direct sample infusion. In the second Screening Stage, the main features seen in the Prec and NL scans are organized into lists of precursor ion/product ion transitions (MRMs) which are then used for the fast, specific, and sensitive interrogation of each individual sample. Data analysis by univariate and multivariate statistical methods is used to identify the most informative MRMs and so classify the individual samples. The compounds (biomarkers) which are responsible for the most informative MRMs in particular sample classes can be investigated in an optional third Identification Stage i.e. in a structural identification study. MRM profiling benefits from the much smaller number of functional groups compared to the number of individual metabolites existing in biological samples (where most metabolites are still unknown), resulting in acquisition of a much smaller data set and a shorter analysis time. The application of MRM Profiling to several biological and clinical problems is used to illustrate its features.
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Lípidos/análisis , Bibliotecas de Moléculas Pequeñas/análisis , Biomarcadores/análisis , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
Evaluation of signaling lipids is essential for measuring biological processes. There is a lack of experimental data regarding the proper storage of extracts for signaling lipid analysis, potentially impacting the procedures that can lead to accurate and reproducible evaluation. In this study, the importance of pre-analytical conditions for analyzing ion transitions for phosphatidylethanolamines (PEs), an abundant signaling phospholipid, was systematically assessed. A novel workflow was utilized involving an MRM-based experimental approach followed by statistical analysis. Specifically, lipids were extracted from the brain, heart, lungs, and serum of C57BL/6 mice. Extract subsets were resuspended in organic solvents prior to storage in various temperature conditions. Mass spectrometry analysis by multiple reaction monitoring (MRM) profiling was performed at four time points (1 day, 2 weeks, 2 months, or 6 months) to measure relative amounts of PEs in distinct lipid extract aliquots. We introduce an innovative statistical workflow to measure the changes in relative amounts of PEs in the profiles over time to determine lipid extract storage conditions in which fewer profile changes occur. Results demonstrated that time is the most significant factor affecting the changes in lipid samples, with temperature and solvent having comparatively minor effects. We conclude that for lipid extracts obtained by Bligh & Dyer extraction, storage at - 80.0 °C without solvent for less than 2 weeks before analysis is ideal. By considering the data generated by this study, lipid extract storage practices may be optimized and standardized, enhancing the validity and reproducibility of lipid assessments.
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Iones , Lípidos/química , Fosfatidiletanolaminas/química , Flujo de Trabajo , Animales , Encéfalo/metabolismo , Lípidos/sangre , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Análisis Multivariante , Miocardio/metabolismo , Fosfolípidos/química , Análisis de Componente Principal , Reproducibilidad de los Resultados , Solventes/química , Temperatura , Distribución TisularRESUMEN
Extracellular vesicles (EVs) convey information used in cell-to-cell interactions. Lipid analysis of EVs remains challenging because of small sample amounts available. Lipid discovery using traditional mass spectrometry platforms based on liquid chromatography and high mass resolution typically employs milligram sample amounts. We report a simple workflow for lipid profiling of EVs based on multiple reaction monitoring (MRM) profiling that uses microgram amounts of sample. After liquid-liquid extraction, individual EV samples were injected directly into the electrospray ionization (ESI) ion source at low flow rates (10 µl/min) and screened for 197 MRM transitions chosen to be a characteristic of several classes of lipids. This choice was based on a discovery experiment, which applied 1,419 MRMs associated with multiple lipid classes to a representative pooled sample. EVs isolated from 12 samples of human lymphocytes and 16 replicates from six different rat cells lines contained an estimated amount of total lipids of 326 to 805 µg. Samples showed profiles that included phosphatidylcholine (PC), sphingomyelin (SM), cholesteryl ester (CE), and ceramide (Cer) lipids, as well as acylcarnitines. The lipid profiles of human lymphocyte EVs were distinguishable using principal component and cluster analysis in terms of prior antibody and drug exposure. Lipid profiles of rat cell lines EV's were distinguishable by their tissue of origin.
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Vesículas Extracelulares/química , Lípidos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Línea Celular , Línea Celular Tumoral , Femenino , Humanos , Lípidos/química , Extracción Líquido-Líquido , Linfocitos/química , Linfocitos/citología , Análisis de Componente Principal , RatasRESUMEN
To assess the effects of acylcarnitine accumulation on muscle insulin sensitivity, a model of muscle acylcarnitine accumulation was generated by deleting carnitine palmitoyltransferase 2 (CPT2) specifically from skeletal muscle (Cpt2Sk-/- mice). CPT2 is an irreplaceable enzyme for mitochondrial long-chain fatty acid oxidation, converting matrix acylcarnitines to acyl-CoAs. Compared with controls, Cpt2Sk-/- muscles do not accumulate anabolic lipids but do accumulate â¼22-fold more long-chain acylcarnitines. High-fat-fed Cpt2Sk-/- mice resist weight gain, adiposity, glucose intolerance, insulin resistance, and impairments in insulin-induced Akt phosphorylation. Obesity resistance of Cpt2Sk-/- mice could be attributed to increases in lipid excretion via feces, GFD15 production, and energy expenditure. L-carnitine supplement intervention lowers acylcarnitines and improves insulin sensitivity independent of muscle mitochondrial fatty acid oxidative capacity. The loss of muscle CPT2 results in a high degree of long-chain acylcarnitine accumulation, simultaneously protecting against diet-induced obesity and insulin resistance.
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Carnitina O-Palmitoiltransferasa/metabolismo , Carnitina/análogos & derivados , Resistencia a la Insulina/fisiología , Obesidad/prevención & control , Animales , Carnitina/metabolismo , Femenino , Humanos , Ratones , Obesidad/tratamiento farmacológicoRESUMEN
This study describes an automated system used for high throughput screening of reaction conditions based on accelerated reactions occurring in small volumes of reagents. Reaction mixtures are prepared in array format using a fluid handling robot and spotted on a flat polytetrafluoroethylene plate at densities up to 6144 per plate. The reaction and analysis steps are performed simultaneously using desorption electrospray ionization (DESI) to release microdroplets containing the reaction mixture from the plate for reaction prior to arrival at a mass spectrometer. Analysis rates are up to 1 reaction mixture per second and data are recorded in real time using an ion trap mass spectrometer. Beacon compounds are used to triangulate position on the plate and this allows tandem mass spectrometry (MS/MS) to be performed on confirm products of interest. Custom software allows the user to control the system. It is also used to receive data from the DESI mass spectrometer to screen the spectra for compounds of interest, to perform MS/MS and to save data. This custom software also communicates with the software controlling the fluid handling robot (Biomek i7) as well as the Beckman software used to prepare reaction mixtures and also the software that controls the solvent used as the DESI spray. Data were recorded for N-alkylation, N-acylation and N-sulfonylation reactions in three 8 hour experiments on successive days to establish the ruggedness and repeatability of the system. Repeatability is high (94-97%) over this period with false negative 6% (depending on noise threshold chosen). Plates containing 384 reaction mixtures are analyzed in 7 min by moving the DESI sprayer in steps under the sprayer instead of continuously.
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Atopic dermatitis (AD) is a multifactorial disease associated with alterations in lipid composition and organization in the epidermis. Multiple variants of AD exist with different outcomes in response to therapies. The evaluation of disease progression and response to treatment are observational assessments with poor inter-observer agreement highlighting the need for molecular markers. SHARPIN-deficient mice (Sharpincpdm) spontaneously develop chronic proliferative dermatitis with features similar to AD in humans. To study the changes in the epidermal lipid-content during disease progression, we tested 72 epidermis samples from three groups (5-, 7-, and 10-weeks old) of cpdm mice and their WT littermates. An agnostic mass-spectrometry strategy for biomarker discovery termed multiple-reaction monitoring (MRM)-profiling was used to detect and monitor 1,030 lipid ions present in the epidermis samples. In order to select the most relevant ions, we utilized a two-tiered filter/wrapper feature-selection strategy. Lipid categories were compressed, and an elastic-net classifier was used to rank and identify the most predictive lipid categories for sex, phenotype, and disease stages of cpdm mice. The model accurately classified the samples based on phospholipids, cholesteryl esters, acylcarnitines, and sphingolipids, demonstrating that disease progression cannot be defined by one single lipid or lipid category.