RESUMO
Platelet ACKR3/CXCR7 surface expression is enhanced and influences prognosis in coronary artery disease (CAD) patients, who exhibit a distinct atherothrombotic platelet lipidome. Current investigation validates the potential of ACKR3/CXCR7 in regulating thromboinflammatory response through its impact on the platelet lipidome. CAD patients with enhanced platelet ACKR3/CXCR7 expression exhibited reduced aggregation. Pharmacological CXCR7 agonist (VUF11207) significantly reduced prothrombotic platelet response in blood from acute coronary syndrome patients ex vivo. CXCR7 agonist administration reduced thrombotic functions and thromboinflammatory plateletleukocyte interactions post-myocardial infarction and arterial injury in vivo. ACKR3/CXCR7 ligation did not affect surface availability of surface receptors, coagulation profile, bleeding time, plasma-dependent thrombin generation (thrombinoscopy), or clot formation (thromboelastography) but counteracted activation-induced phosphatidylserine exposure and procoagulant platelet-assisted thrombin generation. Targeted (micro-UHPLC-ESI-QTrap-MS/MS) and untargeted (UHPLCESI-QTOF-MS/MS) lipidomics analysis revealed that ACKR3/CXCR7 ligation favored generation of antithrombotic lipids (dihomo-γ-linolenic acid [DGLA], 12-hydroxyeicosatrienoic acid [12-HETrE]) over cyclooxygenase-1 (COX-1) or 12-lipoxygenase (12-LOX) metabolized prothrombotic and phospholipase-derived atherogenic lipids in healthy subjects and CAD patients, contrary to antiplatelet therapy. Through 12-HETrE, ACKR3/CXCR7 ligation coordinated with Gαs-coupled prostacyclin receptor to trigger cyclic adenosine monophosphate/protein kinase A-mediated platelet inhibition. ACKR3/CXCR7 ligation reduced generation of lipid agonists and lipid signaling intermediates, which affected calcium mobilization, intracellular signaling, and consequently platelet interaction with physiological matrices and thromboinflammatory secretome. This emphasized its functional dichotomy from prothrombotic CXCR4. Moreover, CXCR7 agonist regulated heparin-induced thrombocytopenia-sera/immunoglobulin G-triggered platelet and neutrophil activation, heparin-induced platelet aggregation, generation of thromboinflammatory lipids, platelet-neutrophil aggregate formation, and thromboinflammatory secretion ex vivo. Therefore, ACKR3/CXCR7 may offer a novel therapeutic strategy in acute/chronic thromboinflammation exaggerated cardiovascular pathologies and CAD.
Assuntos
Receptores CXCR/metabolismo , Trombose , Plaquetas/metabolismo , Humanos , Inflamação/metabolismo , Lipidômica , Lipídeos , Espectrometria de Massas em Tandem , Trombina/metabolismo , Tromboinflamação , Trombose/metabolismoRESUMO
Betulin is a pentacyclic triterpene with demonstrated healing properties in mid-dermal wounds. A few earlier studies have provided insights into the wound healing effects on the molecular level. However, there are still questions left on the molecular targets of betulin. Therefore, a pharmacolipidomics analysis of betulin is undertaken in human immortalized keratinocytes to monitor alterations in the lipid profiles induced by treatment with betulin. For this purpose, lipid extracts of keratinocytes treated with betulin and untreated controls are comprehensively analyzed by an untargeted UHPLC-ESI-QTOF-MS/MS lipidomics profiling workflow using data-independent acquisition. Targeted data processing allows the identification of 611 lipid species from 21 different lipid classes. Statistical analysis of the identified lipids shows significant changes in 440 lipid species that can be described as downregulation of cholesteryl esters and triacylglycerides and upregulation of glycerophospholipids, sphingolipids, and diacylglycerides. Additionally, some other signals corresponding to triterpenes are found in the betulin group and suggested that betulin is incorporated (in the membrane) and metabolized in keratinocytes.
Assuntos
Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Lipidômica/métodos , Espectrometria de Massas em Tandem/métodos , Triterpenos/farmacologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Cicatrização/efeitos dos fármacosRESUMO
Polyunsaturated fatty acids are important signaling molecules. A recent study reported hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid, 12-oxo-5Z,8E,10E-heptadecatrienoic acid, and (12S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid as chemotherapy resistance-inducing factors when tumor cells were treated with cisplatin. Marine-based food supplements like fish oil or algae extracts are rich in polyunsaturated fatty acids and can contain large amounts of hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid. Thus, it was concluded that oral uptake of hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid might induce chemoresistance as shown in a mouse model. Cancer patients tend to consume food supplements containing polyunsaturated fatty acids on a regular basis. The uptake of hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid and (12S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid should be controlled, because even low concentrations of 0.5 ng mL-1 showed chemoresistance-inducing effects in animal experiments. For accurate analysis of hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid and (12S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid a validated method was developed by using ultrahigh-performance liquid chromatography hyphenated to quadrupole time of flight mass spectrometry via electrospray ionization and sample preparation by solid-phase extraction (SPE) with 3-aminopropyl silica. A combined targeted/untargeted approach was utilized using MS/MS by data-independent acquisition with SWATH and applied to commercial food supplements (refined fish oil, fish oil capsules, algae oil capsules, and flaxseed capsules). Accurate quantification of hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid and (12S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid on the MS/MS level with simultaneous untargeted fatty acid screening revealed additional information. The LODs for hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid and (12S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid were 0.036 ng mL-1 and 0.054 ng mL-1, respectively. Since hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid was present in the samples in large amounts and (12S)-hydroxy-5Z,8E,10E-heptadecatrienoic was not expected to be present in high concentrations, two calibration ranges, namely, 0.5-20 ng mL-1 and 5-200 ng mL-1, were validated. An untargeted screening identified 18-39 free fatty acids being present in the lipid extracts of the food supplement samples. Graphical abstract á .
Assuntos
Cromatografia Líquida , Suplementos Nutricionais/análise , Resistencia a Medicamentos Antineoplásicos , Ácidos Graxos/farmacologia , Óleos de Peixe/química , Espectrometria de Massas em Tandem/métodos , Ácidos Graxos/química , Análise de Alimentos , Limite de Detecção , Reprodutibilidade dos TestesRESUMO
Isotope exchange at the histidine C2 atom of imidazole in D2O solution is well known to occur at a significantly slower rate than the exchange of amide protons. Analysis of the kinetics of this isotope-exchange reaction is proposed herein as a method of detecting histidine phosphorylation. This modification of His-containing peptides is challenging to pinpoint because of its instability under acidic conditions as well as during CID-MS analysis. In this work, we investigated the effect of phosphorylation of the histidine side chain in peptides on deuterium-hydrogen exchange (DHX) in the imidazole. The results demonstrate that phosphorylation dramatically slows the rate of the DHX reaction. This phenomenon can be applied to detect phosphorylation of peptides at the histidine residue (e.g., in enzymatic digests). We also found that the influence of the peptide sequence on the exchange kinetics is relatively small. A CID fragmentation experiment revealed that there was no detectable hydrogen scrambling in peptides deuterated at C2 of the imidazole ring. Therefore, MS/MS can be used to directly identify the locations of deuterium ions incorporated into peptides containing multiple histidine moieties.
Assuntos
Histidina/análise , Imidazóis/química , Sequência de Aminoácidos , Deutério/química , Medição da Troca de Deutério/métodos , Hidrogênio/química , Espectrometria de Massas/métodos , Peptídeos/química , FosforilaçãoRESUMO
Endocannabinoids are endogenous lipids with the main function recognized to act as neuromodulators through their cannabinoid receptors. Dysregulation of the endocannabinoid system is implicated in various pathologies, such as inflammatory and neurodegenerative diseases. In this study we describe a sensitive UHPLC-MS/MS method for the analysis of trace levels of 7 endocannabinoids in cerebrospinal fluid samples. The analytes covered comprised 1- and 2-arachidonoylglycerol 1- and 2-AG (which were analysed as sum due to their interconversion), 2-arachidonylglycerol ether 2-AGE, anandamide AEA, N-linoleoyl ethanolamide LEA, N-palmitoyl ethanolamide PEA and N-oleoyl ethanolamide OEA. Analytes were extracted from the biofluid by a simple monophasic procedure involving protein precipitation with acetonitrile (MeCN). The analytical method is based on chromatographic separation of the analytes with solid-core (core-shell, superficially porous) particle column Cortecs C18+ . Gradient elution with changing proportion of water and acetonitrile and constant concentration of formic acid provided reasonable separation of analytes, close elution of analytes and their internal standards and minimized matrix effects in biological samples. For specific detection of the endocannabinoids a triple-quadrupole tandem mass spectrometer with electrospray ionisation (ESI) and selected reaction monitoring (SRM) mode was used, and it provided good assay selectivity. The developed method required a minute volume of the biological samples (50 µL) and achieved excellent sensitivity (the lower limit of detection was between 4.15 and 30.18 pM of the biological sample). Linear calibration was achieved in the range from 25 to 10,545 pM for AEA, 90-3802 pM for 1-AG, 90-724 pM for 2-AG, 12-5226 pM for LEA, 33-13,942 for OEA, 34-23,850 pM for 2-AGE, 72-30,190 for PEA and 10-4218 for AEA-d4 in CSF. The method was validated and revealed relative errors in the range of - 14.7 to + 12.3% at LLOQ and - 14.1 to + 14.2% for the remaining validation range. Precisions were in the acceptable range (< 20% RSD at LLOQ, and <15% for the remaining levels) as well. It was finally used to quantify endocannabinoids in human cerebrospinal fluid obtained from 118 donors. Accurate quantification of endogenous compounds in biological samples was achieved by using two different principal approaches (surrogate matrix for AEA, 2-AG, OEA, 2-AGE, LEA and PEA, and surrogate calibrant for AEA only) and they were evaluated by use of the Passing-Bablok regression. Concentrations (median) of CSF samples of patients suffering from CNS infection and controls were found to be around 160 pM for 1- and 2-AG, 86 pM for AEA, 62 for 2-AGE, 58 for LEA, 93 pM for PEA, and 83 pM for OEA.
Assuntos
Endocanabinoides , Espectrometria de Massas em Tandem , Humanos , Idoso , Pessoa de Meia-Idade , Endocanabinoides/metabolismo , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , AcetonitrilasRESUMO
Lipids play a major role in platelet signaling and activation. In this study, we analyzed the platelet lipidome in an untargeted manner by reversed-phase UHPLC for lipid species separation coupled to high-resolution QTOF-MS/MS in data-independent acquisition (DIA) mode with sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) for compound detection. Lipid identification and peak picking was supported by the characteristic regular elution pattern of lipids differing in carbon and double bond numbers. It was primarily based on post-acquisition targeted feature extraction from the SWATH data. Multiple extracted ion chromatograms (EICs) from SWATH data of diagnostic ions on MS1 and MS2 level from both positive and negative ion mode allowed to distinguish between poorly resolved isomeric lipids based on their distinct fragment ions, which were used for relative quantification at a molecular lipid species level. It supports assay specificity for relative lipid quantitation via multiple quantifiably ions unlike to data-dependent acquisition methods which rely on precursor ions only. This approach was used to analyze human platelet samples. 457 lipids were annotated. Concentrations of lipids were estimated by stable isotope-labelled lipid class-specific internal standards as surrogate calibrants. Heatmaps of lipid concentrations in dependence on carbon and double bond numbers for the distinct lipid classes revealed a snapshot of the platelet lipidome in the resting state with lipid species distributions within classes supporting some functional interpretations. As expected, activation of the platelets by thrombin has led to significant alterations in the platelet lipidome as proven by univariate (volcano plot) and multivariate (PLS-DA) statistics. Several lipids were significantly up-regulated (lysophosphatidylinositols, oxylipins such as thromboxane B2 (TXB2), hydroxyheptadecatrienoic acid (HHT), hydroxyeicosatetraenoic acid (HETE), hydroxyoctadecadienoic acid (HODE), sphingoid-bases, (very) long chain saturated fatty acids) or down-regulated (lysophosphatidylethanolamines, polyunsaturated fatty acids, phosphatidylinositols). Several of them are well known as biomarkers of platelet activation while others may provide some further insights into pathways of platelet activation and platelet metabolism.
Assuntos
Lipidômica , Espectrometria de Massas em Tandem , Plaquetas , Cromatografia Líquida de Alta Pressão , Humanos , TrombinaRESUMO
A new commercially available HPLC column, poly-N-(1H-tetrazole-5-yl)-methacrylamide-bonded stationary phase (Daicel DCpak PTZ), was systematically evaluated for its carbohydrate isomer separation capability by hydrophilic interaction liquid chromatography (HILIC) with charged aerosol detection (CAD) or (tandem) mass spectrometry. Reducing sugars tend to split into two anomer peaks which makes carbohydrate isomer separations in non-derivatized form even more complicated. For practical purposes anomer separations are therefore ideally suppressed which can be accomplished by using high temperature or high pH that are both associated with fast interconversion kinetics leading to peak coalescence, or on the other hand by conditions with low chromatographic anomer selectivity. Four major hexoses (glucose, mannose, galactose, fructose), five main pentoses (ribose, ribulose, xylose, xylulose, arabinose) and five most important disaccharides (maltose, cellobiose, lactose, sucrose, trehalose) were analyzed as single carbohydrate standards by isocratic HILIC with 0.1% (v/v) formic acid and 2 mM ammonium acetate at various temperatures to study anomer interconversion equilibria in a pH-dependent manner. Rate constants of forward (αâß) and backward (ßâα) anomerization and corresponding energy barriers were calculated. The energy barriers of anomerisation were in the range of around 83-91 kJ mol-1 at 298 K and the difference between forward (αâß) and backward reaction (ßâα) was typically between 1-3 kJ mol-1. The systematic studies finally allowed to pick conditions for the simultaneous analysis of all 14 carbohydrates by HILIC-ESI-MS(/MS) with PTZ in gradient elution mode. A combination of carbohydrate isomer-selective LC (with PTZ), tandem MS (with carbohydrate group-selective MS1 and some species-specific SRM transitions) and a simple deconvolution strategy allowed the determination of all carbohydrates of the complex test mixture except for the disaccharide pair lactose and maltose (which can be determined as sum). Consequently, the proposed method represents a successful step towards a global glycometabolomics profiling method of mono- and disaccharides by HILIC-ESI-MS/MS.
Assuntos
Acrilamidas/química , Carboidratos/química , Cromatografia Líquida de Alta Pressão/métodos , Interações Hidrofóbicas e Hidrofílicas , Tetrazóis/química , Carboidratos/isolamento & purificação , Meia-Vida , Isomerismo , Cinética , Padrões de Referência , Temperatura , TermodinâmicaRESUMO
A Poly(N-(1H-tetrazole-5-yl)methacrylamide)-bonded (PTZ) stationary phase has recently gained increased attention in hydrophilic liquid chromatography (HILIC) mode for separation of polar compounds and is now commercially available as DCpak PTZ. It is chromatographically characterized in this work. The property of the new column was proven to be the most hydrophilic and acidic by separation of mixtures of purines and pyrimidines (theophylline, theobromine, uridine and 2'-deoxyuridine) in comparison to other two commercial columns Luna HILIC and LiChrospher Diol column. The retention mechanism of the new column was investigated by design-of-experiment (DoE) approach using factorial design models. Water/acetonitrile ratio in the mobile phase, buffer salt concentration and buffer pH were considered factors employing the purine/pyrimidine mixture and glucose derivatives as test samples. The resultant retention model coefficients and contour plots documented the complementary retention and selectivity profiles of the new PTZ column as compared to Diol and Luna HILIC. Moreover, it became clearly evident that the PTZ column exhibits its best HILIC performance at eluent pH ≥ 5 because the NH group in tetrazolyl moiety is dissociated under these conditions. The applicability and great potential of the new HILIC column was proven by the chromatographic separation of complex mixtures of very hydrophilic glucose and glucose derivatives (sucrose, glucosamine, glucuronic acid, glucose-1-phosphate and trehalose, glucose, maltose, glucosamine-6-phosphate, glucose-6-phosphate and gluconic acid δ-lactone) as well as monosaccharides found in N-glycans. It is concluded that the new DCpak PTZ HILIC column could have good prospects for the separation of polar compounds e.g. in metabolomics and glycomics.
Assuntos
Cromatografia Líquida/métodos , Interações Hidrofóbicas e Hidrofílicas , Tetrazóis/química , Glucose/análise , Glicoproteínas/análiseRESUMO
In this study, a combined targeted/untargeted UHPLC-ESI-QTOF-MS/MS method for the targeted quantitative analysis of the primary platelet lipid mediators thromboxane B2 (TXB2), 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid (HHT) and its oxidation product 12-keto-5Z,8E,10E-heptadecatrienoic acid (KHT) was developed, which allowed simultaneous untargeted profiling for the detection of other lipid biomarkers such as other oxylipins and fatty acids (FAs) in platelet releasates. A general procedure for the synthesis of keto-analogs from hydroxylated polyunsaturated FAs (PUFAs) using Dess-Martin periodinane oxidation reagent was proposed for the preparation of KHT standard. MS detection was performed in data independent acquisition (DIA) mode with sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) in the range of 50-500â¯Da with variable window sizes. The LC-MS/MS assay was validated for the targeted analytes and applied for analysis of supernatants derived from resting platelets and from platelets treated with thrombin. The targeted analytes KHT, HHT and TXB2 were found at highly elevated levels in the activated platelet releasates. On average, 13⯱â¯7, 15⯱â¯9, and 0.6⯱â¯0.2 attomols per platelet were released upon thrombin-activation. Furthermore, the simultaneous untargeted profiling (nâ¯=â¯8 in each group) revealed that these oxylipins are released with a pool of other (significantly upregulated) oxidized (12-HETE, 12-HEPE) and non-oxidized PUFAs. All these compounds can be considered additional biomarkers of platelet activation complementing the primary platelet activation marker thromboxane B2. The other lipids may support platelet activation or trigger other biological actions with some potential implications in thromboinflammation.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ácidos Graxos/sangue , Ativação Plaquetária/efeitos dos fármacos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Trombina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Ácidos Graxos Insaturados/sangue , Ácidos Graxos Insaturados/síntese química , Humanos , Limite de Detecção , Tromboxano B2/sangueRESUMO
Oxylipins, the oxidation products of polyunsaturated fatty acids, are important signaling molecules in living organisms. Some of them have pro-inflammatory properties, while others act as pro-resolving agents. Oxylipins also play a major role in platelet biology and the progression of thrombo-inflammation. Depending on their structure, they may be pro-thrombotic or anti-thrombotic. For an unbiased biological interpretation, a detailed analysis of a broad spectrum of oxylipins including their stereoisomers is necessary. In our work, we developed for the first time an enantioselective UHPLC-ESI-MS/MS assay which allows quantifying individual oxylipin enantiomers. The assay made use of a sub-2µm particle-based amylose-(3,5-dimethylphenylcarbamate) chiral stationary phase (Chiralpak IA-U) under MS-compatible reversed-phase conditions. It covered 19 enantiomeric pairs of oxylipins and one diasteromeric pair of a lipid mediator: 2 pairs of hydroxyoctadecadienoic acids (HODE), 6 pairs of hydroxyeicosatetraenoic acids (HETE), 5 pairs of hydroxyeicosapentaenoic acids (HEPE), 3 pairs of hydroxydocosahexaenoic acids (HDoHE) and one pair of each: resolvins D1, hydroxyeicosatrienoic acid (HETrE), hydroxyoctadecatrienoic acid (HOTrE) and dihydroxyeicosatetraenoic acid (DiHETE). The new method is fast and showed outstanding peak resolution for most of the isomeric pairs. Excellent method sensitivity (average LOD was equal to 2.7 pg on column) was obtained by using a triple quadrupole instrument as a detector in a targeted, selected reaction monitoring (SRM) mode. The applicability of the method was verified by preliminary validation. It was then applied to analyze oxylipins produced by autoxidation of polyunsaturated fatty acids (PUFA) in air. Multiple oxylipins were found in each of the samples as racemic mixtures and served as reference substances for identification. Finally, the new enantioselective UHPLC method was applied to analyze releasates from platelets in resting state, and following activation with thrombin. The highest abundant oxylipin in the platelet releasate was 12(S)-HETE, but many other oxylipins were found in the thrombin activated samples, usually as single enantiomers (e.g. 12(S)-HEPE, 11(R)-HETE, 9(R)-HODE, 13-(S)-HODE, 14(S)-HDoHE). The latter was detected at about similar concentration in resting platelet releasates as well. 15-HETE showed elevated levels for both R-and S-enantiomers in releasates of thrombin-activated platelets. 12-HETrE was found presumably as both enantiomers, however, retention time inconsistencies indicate that the R-enantiomer is actually a different compound, maybe another constitutional isomer with different double-bond configuration.
Assuntos
Plaquetas/química , Cromatografia Líquida de Alta Pressão/métodos , Oxilipinas/análise , Polissacarídeos/química , Plaquetas/metabolismo , Ácidos Graxos Insaturados/análise , Ácidos Graxos Insaturados/química , Humanos , Limite de Detecção , Oxirredução , Estereoisomerismo , Espectrometria de Massas em TandemRESUMO
Oxylipins play an important role in cell signaling and they act as auto- and paracrine factors. There are numerous reports on the analysis of oxylipins in biofluids, especially in plasma. Only a limited number of studies addressed the analysis of oxylipins in platelets using modern, sensitive LCMS methods, even though these compounds have a huge impact on platelet functions and thrombo-inflammation. In this work, a new method based on superficially porous particle (2.7⯵m) capillary column (0.5â¯mm ID) and micro-liquid chromatography coupled to tandem mass spectrometry (µUHPLC-ESI-QqQ-MS/MS) method has been developed, optimized and validated. It has finally been successfully applied for human plasma and platelet analysis. The method allows the precise and accurate simultaneous quantification of 42 oxylipins with 13 deuterated internal standards. Solid phase extraction with Bond Elut Certify II provides good extraction recoveries (on average around 75 %). The µUHPLC-MS/MS method is selective, sensitive (LOQs between 30 and 150â¯pg/mL) and shows good linearity. Limits of detections for most of the compounds are between 2 and 250â¯fmol on column. Twenty-three oxylipins have been detected in plasma and 19 in non-activated (resting) platelets (all samples were from healthy donors). The µUHPLC-MS/MS method uses very low volume of mobile phase (less than 250⯵L of organic solvents in mobile phase per analysis), and therefore is considered environmentally friendly. It also turned out to be robust enough for routine analysis.
Assuntos
Oxilipinas , Espectrometria de Massas em Tandem , Plaquetas , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , HumanosRESUMO
Self-assembly is a widely studied, spontaneous, and reversible phenomenon leading to the formation of the ordered structures by non-covalent specific interactions among starting molecules. In this work, a new template for the self-assembly of polypeptides based on peptides containing the C-terminal histidinehydroxamic acid moiety and Cu(2+) ions is characterized. Two peptide (tripeptide and pentadecapeptide) hydroxamic acid systems were synthesized and their interactions with Cu(2+) ions were investigated, revealing a high stability of the supramolecular assemblies formed. The supramolecular metallacrown-based L4Cu5 complexes exist at physiological pH in the presence of Cu(2+) ions as is evidenced from the spectroscopic methods, ESI mass spectrometry, and physicochemical techniques.