RESUMEN
Prior MALDI mass spectrometry imaging (MALDI-MSI) studies reported significant changes in phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs), and sphingomyelins (SMs) in ischemic rat brains yet overlooked the information on other classes of PLs and SLs and provided very little or no validation on the detected lipid markers. Relative quantitation of four classes of PLs and two classes of SLs in the ischemic and normal temporal cortex (TCX), parietal cortex (PCX), and striatum (ST) of rats was performed with hydrophilic interaction chromatography (HILIC)-tandem mass spectrometry (MS/MS) analyses, and the marker lipid species was identified by multivariate data analysis and validated with additional tissue cohorts. The acquired lipid information was sufficient in differentiating individual anatomical regions under different pathological states, identifying region-specific ischemic brain lipid markers and revealing additional PL and SL markers not reported previously. Validation of orthogonal partial least square discriminating analysis (OPLS-DA) identified ischemic brain lipid markers yielded much higher classification accuracy, precision, specificity, sensitivity, and lower false positive and false negative rates than those from the volcano plot analyses using conventional statistical significance and a fold change of two as the cutoff and provided a wider prospective to ischemia-associated brain lipid changes.
RESUMEN
Lipids perform multiple biological functions and reflect the physiology and pathology of cells, tissues, and organs. Here, we sought to understand lipid content in relation to tumor pathology by characterizing phospholipids and sphingolipids in the orthotopic mouse glioma using MALDI MS imaging (MSI) and LC-MS/MS. Unsupervised clustering analysis of the MALDI-MSI data segmented the coronal tumoral brain section into 10 histopathologically salient regions. Heterogeneous decrease of the common saturated phosphatidylcholines (PCs) in the tumor was accompanied by the increase of analogous PCs with one or two additional fatty acyl double bonds and increased lyso-PCs. Polyunsaturated fatty acyl-PCs and ether PCs highlighted the striatal tumor margins, whereas the distributions of other PCs differentiated the cortical and striatal tumor parenchyma. We detected a reduction of SM d18:1/18:0 and the heterogeneous mild increase of SM d18:1/16:0 in the tumor, whereas ceramides accumulated only in a small patch deep in the tumoral parenchyma. LC-MS/MS analyses of phospholipids and sphingolipids complemented the MALDI-MSI observation, providing a snapshot of these lipids in the tumor. Finally, the proposed mechanisms responsible for the tumoral lipid changes were contrasted with our interrogation of gene expression in human glioma. Together, these lipidomic results unveil the aberrant and heterogeneous lipid metabolism in mouse glioma where multiple lipid-associated signaling pathways underline the tumor features, promote the survival, growth, proliferation, and invasion of different tumor cell populations, and implicate the management strategy of a multiple-target approach for glioma and related brain malignancies.
Asunto(s)
Glioma , Metabolismo de los Lípidos , Ratones , Humanos , Animales , Cromatografía Liquida , Lipidómica , Espectrometría de Masas en Tándem , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Fosfolípidos , Esfingolípidos/análisisRESUMEN
Phospholipids (PLs) and sphingolipids (SLs) perform critical structural and biological functions in cells. The structure of these lipids, including the stereospecificity and double-bond position of fatty acyl (FA) chains, is critical in decoding lipid biology. In this study, we presented a simple in-source fragmentation (ISF) MALDI/TOF mass spectrometry method that affords complete structural characterization of PL and SL molecules. We analyzed several representative unsaturated lipid species including phosphatidylcholine (PC), plasmalogen PC (pPC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), cardiolipin (CL), sphingomyelin (SM), and ceramide (Cer). Fragment ions reflecting the FA chains at sn-1 and sn-2 position, and those characteristics of the head groups of different PL classes, are readily identified. Specific fragment ions from cleavages of the C-C bond immediately adjacent to the cis C=C double-bond position(s) of FA chains and the trans C=C double bond of the sphingosine constituents allow precise localization of double bonds. The identities of the exemplary product ions from vinylic, allylic, and double-bond cleavages were also verified by LIFT-TOF/TOF. Identification of individual PL species in the lipid mixture was also carried out with ISF-MALDI/TOF. Together, this approach provides a simple yet effective method for structural characterization of PLs and SLs without the additional modification on the instrument hardware, and serves as a simple tool for the identification of lipids.
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Fosfolípidos , Esfingolípidos , Ceramidas , Fosfatidilcolinas , Fosfolípidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Esfingolípidos/análisisRESUMEN
Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) of lipids is considered one of the shotgun lipidomic techniques that explores the in situ distribution of lipids in tissue sections. To successfully perform this task, knowledge and experience in the conventional cryosection, tissue section collection and handling, and mass spectrometry data analysis and signal processing are needed. A MALDI-MSI protocol covering from the fresh organ collection, cryosection and tissue processing, matrix application, MSI data acquisition, to the final MSI display of lipid distribution in the brain section of an ischemic stroke rat is described to exemplify this technique. Due to the multidisciplinary nature of this approach, plenty of preparation, practice, and familiarization to several critical steps ahead of the engagement with actual biological samples are needed to ensure a successful MALDI-MSI presentation of lipids in situ.
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Isquemia Encefálica/diagnóstico por imagen , Lipidómica/métodos , Lípidos/análisis , Animales , Química Encefálica , Isquemia Encefálica/metabolismo , Criopreservación , Imagen Molecular , Ratas , Programas Informáticos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Conservación de TejidoRESUMEN
Uncommon lipids in biotechnologically important Corynebacterium glutamicum and pathogen Corynebacterium striatum in genus Corynebacterium are isolated and identified by linear ion-trap multiple stage mass spectrometry (LIT MSn) with high resolution mass measurement. We redefined several lipid structures that were previously mis-assigned or not defined, including cytidine diphosphate diacylglycerol (CDP-DAG), glucuronosyl diacylglycerol (GlcA-DAG), (α-d-mannopyranosyl)-(1 â 4)-(α-D-glucuronyl diacyglycerol (Man-GlcA-DAG), 1-mycolyl-2-acyl-phosphatidylglycerol (MA-PG), acyl trehalose monomycolate (acyl-TMM). We also report the structures of mycolic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, trehalose dimycolate lipids in which many isomeric structures are present. The LIT MSn approaches afford identification of the functional group, the fatty acid substituents and their regiospecificity in the molecules, revealing the biodiversities of the lipid species in two Corynebacterium strains that have played very different and important roles in human nutrition and health.
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Corynebacterium glutamicum/química , Corynebacterium/química , Lípidos/química , Lípidos/aislamiento & purificación , Factores Cordón/química , Diglicéridos/química , Humanos , Metabolismo de los Lípidos , Lípidos/clasificación , Fosfatidilgliceroles/química , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Fatty acyl coenzyme A esters (FA-CoAs) are important crossroad intermediates in lipid catabolism and anabolism, and the structures are complicated. Several mass spectrometric approaches have been previously described to elucidate their structures. However, a direct mass spectrometric approach toward a complete identification of the molecule, including the location of unsaturated bond(s) in the fatty acid chain has not been reported. In this study, we applied a simple MALDI/TOF mass spectrometric method to a near-complete characterization of long-chain FA-CoAs, including the location(s) of the double bond in the fatty acyl chain, and the common structural features that recognize FA-CoAs. Negative ion mass spectra of saturated, monounsaturated, and polyunsaturated FA-CoAs were acquired with a MALDI/TOF mass spectrometer using 2,5-dihydroxybenzoic acid as the matrix and ionized with a laser fluence two folds of the threshold to induce the in-source fragmentation (ISF) of the analytes. The resulting ISF spectra contained fragment ions arising from specific cleavages of the C-C bond immediate adjacent to the acyl double-bond. This structural feature affords locating the double-bond position(s) of the fatty acyl substituent. Thereby, positional isomer such as 18:3(n - 3) and 18:3(n - 6) FA-CoA can be differentiated without applying tandem mass spectrometry.
RESUMEN
Focused ultrasound (FUS) exposure with the presence of microbubbles has been shown to transiently open the blood-brain barrier (BBB), and thus has potential to enhance the delivery of various kinds of therapeutic agents into brain tumors. The purpose of this study was to assess the preclinical therapeutic efficacy of FUS-BBB opening for enhanced temozolomide (TMZ) delivery in glioma treatment. FUS exposure with microbubbles was delivered to open the BBB of nude mice that were either normal or implanted with U87 human glioma cells. Different TMZ dose regimens were tested, ranging from 2.5 to 25 mg/kg. Plasma and brain samples were obtained at different time-points ranging from 0.5 to 4 hours, and the TMZ concentration within samples was quantitated via a developed LC-MS/MS procedure. Tumor progression was followed with T2-MRI, and animal survival and brain tissue histology were conducted. Results demonstrated that FUS-BBB opening caused the local TMZ accumulation in the brain to increase from 6.98 to 19 ng/mg. TMZ degradation time in the tumor core was found to increase from 1.02 to 1.56 hours. Improved tumor progression and animal survival were found at different TMZ doses (up to 15% and 30%, respectively). In conclusion, this study provides preclinical evidence that FUS-BBB opening increases the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting the potential for clinical application to improve current brain tumor treatment.
Asunto(s)
Antineoplásicos/farmacología , Barrera Hematoencefálica/metabolismo , Dacarbazina/análogos & derivados , Sistemas de Liberación de Medicamentos/métodos , Glioma/tratamiento farmacológico , Ultrasonido , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Barrera Hematoencefálica/efectos de los fármacos , Capilares/efectos de los fármacos , Capilares/patología , Permeabilidad Capilar , Línea Celular Tumoral , Dacarbazina/administración & dosificación , Dacarbazina/metabolismo , Dacarbazina/farmacología , Dacarbazina/uso terapéutico , Glioma/irrigación sanguínea , Glioma/metabolismo , Glioma/patología , Humanos , Masculino , Ratones , Microburbujas , Análisis de Supervivencia , Temozolomida , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND AND OBJECTIVES: It is well established by a large number of randomized controlled trials that lowering blood pressure (BP) and low-density lipoprotein cholesterol (LDL-C) by drugs are powerful means to reduce stroke incidence, but the optimal BP and LDL-C levels to be achieved are largely uncertain. Concerning BP targets, two hypotheses are being confronted: first, the lower the BP, the better the treatment outcome, and second, the hypothesis that too low BP values are accompanied by a lower benefit and even higher risk. It is also unknown whether BP lowering and LDL-C lowering have additive beneficial effects for the primary and secondary prevention of stroke, and whether these treatments can prevent cognitive decline after stroke. RESULTS: A review of existing data from randomized controlled trials confirms that solid evidence on optimal BP and LDL-C targets is missing, possible interactions between BP and LDL-C lowering treatments have never been directly investigated, and evidence in favour of a beneficial effect of BP or LDL-C lowering on cognitive decline is, at best, very weak. CONCLUSION: A new, large randomized controlled trial is needed to determine the optimal level of BP and LDL-C for the prevention of recurrent stroke and cognitive decline.
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LDL-Colesterol/sangre , Trastornos del Conocimiento/prevención & control , Hipercolesterolemia/tratamiento farmacológico , Accidente Cerebrovascular/prevención & control , Presión Sanguínea/efectos de los fármacos , Colesterol , Cognición , Humanos , Masculino , Prevención Primaria , Ensayos Clínicos Controlados Aleatorios como Asunto , Recurrencia , Prevención SecundariaRESUMEN
BACKGROUND AND OBJECTIVES: The SBP values to be achieved by antihypertensive therapy in order to maximize reduction of cardiovascular outcomes are unknown; neither is it clear whether in patients with a previous cardiovascular event, the optimal values are lower than in the low-to-moderate risk hypertensive patients, or a more cautious blood pressure (BP) reduction should be obtained. Because of the uncertainty whether 'the lower the better' or the 'J-curve' hypothesis is correct, the European Society of Hypertension and the Chinese Hypertension League have promoted a randomized trial comparing antihypertensive treatment strategies aiming at three different SBP targets in hypertensive patients with a recent stroke or transient ischaemic attack. As the optimal level of low-density lipoprotein cholesterol (LDL-C) level is also unknown in these patients, LDL-C-lowering has been included in the design. PROTOCOL DESIGN: The European Society of Hypertension-Chinese Hypertension League Stroke in Hypertension Optimal Treatment trial is a prospective multinational, randomized trial with a 3â×â2 factorial design comparing: three different SBP targets (1, <145-135; 2, <135-125; 3, <125â mmHg); two different LDL-C targets (target A, 2.8-1.8; target B, <1.8 âmmol/l). The trial is to be conducted on 7500 patients aged at least 65 years (2500 in Europe, 5000 in China) with hypertension and a stroke or transient ischaemic attack 1-6 months before randomization. Antihypertensive and statin treatments will be initiated or modified using suitable registered agents chosen by the investigators, in order to maintain patients within the randomized SBP and LDL-C windows. All patients will be followed up every 3 months for BP and every 6 months for LDL-C. Ambulatory BP will be measured yearly. OUTCOMES: Primary outcome is time to stroke (fatal and non-fatal). Important secondary outcomes are: time to first major cardiovascular event; cognitive decline (Montreal Cognitive Assessment) and dementia. All major outcomes will be adjudicated by committees blind to randomized allocation. A Data and Safety Monitoring Board has open access to data and can recommend trial interruption for safety. SAMPLE SIZE CALCULATION: It has been calculated that 925 patients would reach the primary outcome after a mean 4-year follow-up, and this should provide at least 80% power to detect a 25% stroke difference between SBP targets and a 20% difference between LDL-C targets.
Asunto(s)
Trastornos del Conocimiento/prevención & control , Hipertensión/complicaciones , Prevención Secundaria/métodos , Accidente Cerebrovascular/prevención & control , Anciano , Antihipertensivos/uso terapéutico , Presión Sanguínea/efectos de los fármacos , Determinación de la Presión Sanguínea , China , LDL-Colesterol/sangre , Cognición , Demencia/etiología , Europa (Continente) , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Hipertensión/tratamiento farmacológico , Ataque Isquémico Transitorio/tratamiento farmacológico , Masculino , Estudios Prospectivos , RecurrenciaRESUMEN
Cardiolipin (CL) is a class of phospholipid tightly associated with the mitochondria functions and a prime target of oxidative stress. Peroxidation of CL dissociates its bound cytochrome C, a phenomenon that reflects oxidative stress sustained by the organ and a trigger for the intrinsic apoptotic pathway. However, CL distribution in normal organ tissues has yet to be documented. Fresh rat organs were snap-frozen, cut into cryosections that were subsequently desalted with ammonium acetate solution, and vacuum-dried. CL distribution in situ was determined using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique on sections sublimed with 2,5-dihydroxybenzoic acid. CL images in rat cardiac ventricular section showed a homogeneous distribution of a single m/z 1447.9 ion species that was confirmed as the (18:2)4 CL by tandem mass spectrometry. The presence of low abundant (18:2)3(18:1) CL with the bulk (18:2)4 CL in quadriceps femoris rendered the muscle CL exhibiting a slightly deviated isotopic pattern from that of cardiac muscle. In rat liver, MALDI-MSI unveiled three CL-containing mass ranges, each with a unique in situ distribution pattern. Co-registration of the CL ion images with its stained liver section image further revealed the association of CLs in each mass range with the functional zones in the liver parenchyma and suggests the participation of in situ CLs with localized hepatic functions such as oxidation, conjugation, and detoxification. The advances in CL imaging offer an approach with molecular accuracy to reveal potentially dysregulated metabolic machineries in acute and chronic diseased states.
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Cardiolipinas/análisis , Ventrículos Cardíacos/ultraestructura , Músculo Esquelético/ultraestructura , Miocardio/ultraestructura , Animales , Cardiolipinas/metabolismo , Gentisatos/química , Ventrículos Cardíacos/química , Hígado/química , Hígado/ultraestructura , Masculino , Microtomía , Imagen Molecular/instrumentación , Imagen Molecular/métodos , Músculo Esquelético/química , Miocardio/química , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Dysregulations of RNA A-to-I editing are associated with developmental defects in mouse and human diseases. Although several methods of identifying RNA A-to-I editing sites are currently available, most of the critical editing targets responsible for the important biological functions of adenosine deaminases that act on RNA (ADARs) remain unknown. Here we report a modified I-specific cleavage method that improves the quality of the RNA product. Preliminary microarray comparison of RNAs subjected to I-specific cleavage or mock digestion reported 165 genes that showed more than 0.2-fold reductions due to the cleavage. Six of the 165 genes were randomly selected for further verification, and three were verified to be targets of I-specific cleavage. This method may provide an alternative method of identifying novel RNA A-to-I editing sites using a microarray and facilitate the inquiry into the roles of RNA A-to-I editing in various biological processes.
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Inosina/química , División del ARN , Edición de ARN , Adenosina Desaminasa/química , Animales , Química Encefálica , Ratones , Ratones Endogámicos ICR , Análisis por MicromatricesRESUMEN
The purpose of this study is to assess the preclinical therapeutic efficacy of magnetic resonance imaging (MRI)-monitored focused ultrasound (FUS)-induced blood-brain barrier (BBB) disruption to enhance Temozolomide (TMZ) delivery for improving Glioblastoma Multiforme (GBM) treatment. MRI-monitored FUS with microbubbles was used to transcranially disrupt the BBB in brains of Fisher rats implanted with 9L glioma cells. FUS-BBB opening was spectrophotometrically determined by leakage of dyes into the brain, and TMZ was quantitated in cerebrospinal fluid (CSF) and plasma by LC-MS\MS. The effects of treatment on tumor progression (by MRI), animal survival and brain tissue histology were investigated. Results demonstrated that FUS-BBB opening increased the local accumulation of dyes in brain parenchyma by 3.8-/2.1-fold in normal/tumor tissues. Compared to TMZ alone, combined FUS treatment increased the TMZ CSF/plasma ratio from 22.7% to 38.6%, reduced the 7-day tumor progression ratio from 24.03 to 5.06, and extended the median survival from 20 to 23 days. In conclusion, this study provided preclinical evidence that FUS BBB-opening increased the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting its clinical potential for improving current brain tumor treatment.
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Antineoplásicos Alquilantes/uso terapéutico , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de la radiación , Neoplasias Encefálicas/tratamiento farmacológico , Dacarbazina/análogos & derivados , Glioblastoma/tratamiento farmacológico , Sonido , Animales , Antineoplásicos Alquilantes/farmacocinética , Encéfalo/patología , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/mortalidad , Línea Celular Tumoral , Dacarbazina/farmacocinética , Dacarbazina/uso terapéutico , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Glioblastoma/diagnóstico , Glioblastoma/mortalidad , Imagen por Resonancia Magnética , Masculino , Ratas , TemozolomidaRESUMEN
AIMS: Proliferation and migration of vascular smooth muscle cells (VSMCs) can cause atherosclerosis and neointimal formation. MicroRNAs have been shown to regulate cell proliferation and phenotype transformation. We discovered abundant expression of microRNA-195 in VSMCs and conducted a series of studies to identify its function in the cardiovascular system. METHODS AND RESULTS: MicroRNA-195 expression was initially found to be altered when VSMCs were treated with oxidized low-density lipoprotein (oxLDL) in a non-replicated microRNA array experiment. Using cellular studies, we found that microRNA-195 reduced VSMC proliferation, migration, and synthesis of IL-1ß, IL-6, and IL-8. Using bioinformatics prediction and experimental studies, we showed that microRNA-195 could repress the expression of Cdc42, CCND1, and FGF1 genes. Using a rat model, we found that the microRNA-195 gene, introduced by adenovirus, substantially reduced neointimal formation in a balloon-injured carotid artery. In situ hybridization confirmed the presence of microRNA-195 in the treated arteries but not in control arteries. Immunohistochemistry experiments showed abundant Cdc42 in the neointima of treated arteries. CONCLUSIONS: We showed that microRNA-195 plays a role in the cardiovascular system by inhibiting VSMC proliferation, migration, and proinflammatory biomarkers. MicroRNA-195 may have the potential to reduce neointimal formation in patients receiving stenting or angioplasty.
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Traumatismos de las Arterias Carótidas/terapia , Terapia Genética , MicroARNs/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima , Adenoviridae/genética , Animales , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Movimiento Celular , Proliferación Celular , Células Cultivadas , Biología Computacional , Ciclina D1/genética , Ciclina D1/metabolismo , Modelos Animales de Enfermedad , Factor 1 de Crecimiento de Fibroblastos/genética , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Inmunohistoquímica , Hibridación in Situ , Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipoproteínas LDL/metabolismo , Masculino , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Transfección , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Ischemia-mediated lipidomic changes in rat brains were explored by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) profiling and imaging after in situ desalting which drastically simplified the spectral presentation of tissue lipids. Removal of interference from the massively changed cations in response to tissue damage permitted the revelation of subtle yet important lipidomic changes. The identities of the detected lipids were confirmed by MALDI tandem mass spectrometry (MALDI-MS/MS). The MALDI-MS imaging (MALDI-MSI) result of lysophosphatidylcholine 16:0 (LPC 16:0) in the desalted brain section appeared essentially identical to that of sodiated LPC 16:0 in the adjacent undesalted section and verified the suitability of the desalting method for the MALDI-MSI studies of lipids in tissue. Other than the consistently decreased phosphatidylcholine (PC) 16:0/18:1, images of PCs containing all saturated, or combined saturated and monounsaturated fatty acyl (MUFA) residues revealed their parenchymal increase by ischemia. Images of PCs containing polyunsaturated fatty acyl (PUFA) residues in normal cortex showed laminated patterns similar to cortical lamina. Ischemia reduced the abundance of PC 16:0/20:4 and PC 16:0/22:6 and disrupted the laminated distribution of the former. However, ischemia increased the subcortical abundance of PUFA-PCs containing stearoyl residue and confined their cortical increase within limited areas. Image of parenchymal sphingomyelin 18:0 (SM 18:0) showed its consistent decrease by ischemia that paralleled the increase of ceramide 18:0-H(2)O in region of moderate to high SM abundance. The above results presented the lipidomic changes largely different from previous MALDI-MSI results and suggested a window of intervention that may benefit the management of cerebrovascular accident and other brain injuries.
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Química Encefálica , Isquemia/metabolismo , Metabolismo de los Lípidos , Lípidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Encéfalo/metabolismo , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Direct MALDI-mass spectrometry (MALDI-MS) profiling of tissue lipids often observes isobaric phosphatidylcholine (PC) species caused by the endogenous alkali metal ions that bias the relative abundance of tissue lipids. Fresh rat brain cryosections were washed with 70% ethanol (EtOH), water (H2O), or 150 mM ammonium acetate (NH4Ac), and the desalting effectiveness of each fluid was evaluated by MALDI-MS profiling of PC and sphingomyelin (SM) species in tissue and in the washing runoff. The results indicated that EtOH and H2O only partially desalted the tissue lipids, yet both substantially displaced the tissue lipids to the washing runoffs. On the other hand, NH4Ac effectively desalted the tissue lipids and produced a runoff containing no detectable PCs or SMs. NH4Ac wash also unveiled the underlying changes of PCs and SMs in the infarcted rat cortex previously masked by edema-caused increase of tissue sodium. The MS/MS of an isobaric PC in the infarcted cortex revealed the precursor change as the result of NH4Ac wash and confirmed the desalting effectiveness of such wash. Other than desalting, NH4Ac wash also removes contaminants in tissue, enhances the overall spectral quality, and benefits additionally in profiling of biological molecules in tissue.
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Lípidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Técnicas In Vitro , Masculino , Fosfatidilcolinas/análisis , Ratas , Ratas Sprague-Dawley , Esfingomielinas/análisisRESUMEN
Stroke, a deleterious cerebrovascular event, is caused by a critical reduction in the blood flow to the brain parenchyma that leads to brain injury and loss of brain functions. The inflammatory responses following ischemia often aggravate the neurological damage. Several pro-inflammatory mediators released after stroke are closely related to the metabolism of phospholipids. In this study we directly profiled the changes in phospholipids in the infarcted rat cerebral cortex 24 hours after middle cerebral artery occlusion (MCAO) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Several phosphatidylcholine (PC) species and sphingomyelin (SM) were significantly decreased after infarction. The cationization pattern of the remaining PCs showed a prominent shift from a mostly potassiated or protonated form to a predominantly sodiated pattern. Stroke also elevated the lysophosphatidylcholines (LPCs) and heme in tissue. The isobaric pairs in PC and LPC classes were resolved by masses through their respective alkali metal adducts in the presence of CsCl. The major fatty acyl LPC species were also structurally confirmed by MALDI-MS/MS. Overall, the results described the changes in PC and LPC species in the infarcted rat cortex. The elevated tissue levels of LPCs and heme signify the ongoing pathological lipid breakdown and the state of parenchymal inflammation. The elevated LPC level in tissue suggests a means of intervention through lysophospholipid metabolism that could potentially benefit the management of stroke and other acute neurological injuries.
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Isquemia Encefálica/complicaciones , Encéfalo/metabolismo , Lisofosfolípidos/metabolismo , Fosfolípidos/metabolismo , Accidente Cerebrovascular/metabolismo , Animales , Química Encefálica , Modelos Animales de Enfermedad , Humanos , Lisofosfolípidos/análisis , Masculino , Fosfolípidos/análisis , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Accidente Cerebrovascular/etiologíaRESUMEN
Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool that has allowed researchers to directly probe tissue molecular structure and drug content with minimal manipulations, while maintaining anatomical integrity. In the present work glycerophospholipids and sphingolipids images were acquired from 16 µm thick coronal rat brain sections using MALDI-MS. Images of phosphatidylinositol 38:4 (PI 38:4), suifatide 24:1 (ST 24:1), and hydroxyl sulfatide 24:1 (ST 24:1 (OH)) were acquired in negative ion mode, while the images of phosphatidylcholine 34:1 (PC 34:1), potassiated phosphatidylcholines 32:0 (PC32:0 + K(+)) and 36:1 (PC 36:1 +K(+)) were acquired in positive ion mode. The images of PI 38:4 and PC 36:1+K(+) show the preferential distribution of these two lipids in gray matter; and the images of two sulfatides and PC 32:0+K(+) show their preferential distribution in white matter. In addition, the gray cortical band and its adjacent anatomical structures were also identified by contrasting their lipid makeup. The resulting images were compared to lipid images acquired by secondary ion mass spectrometry (SIMS). The suitability of TLC sprayers, Collison Nebulizer, and artistic airbrush were also evaluated as means for matrix deposition.
RESUMEN
The lipid membrane is the portal to the cell and its first line of defense against the outside world. Its plasticity, diversity and powers of accommodation in a myriad of environments, mirrored by the varied make up of the cells it protects, are unparalleled. Glycerophospholipids are one of its major components. In cell membranes the extracellular layer is mainly made up of positively charged glycolipids, while the intracellular one's main components are negatively charged. Advances in mass spectrometry have allowed the direct probing of tissues, and thus a direct approach to probing membranes make up was developed. Until recently most studies have focused on proteins. An overview of the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) for the direct analysis of phospholipids in various tissue is presented. Molecular ions corresponding to phosphatidylcholines, sphingomyelin, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols and sulfatides were mapped.
Asunto(s)
Glicerofosfolípidos/metabolismo , Glicoesfingolípidos/metabolismo , Animales , Masculino , Especificidad de Órganos , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Tyrosine sulfation is a post-translational modification entailing covalent attachment of sulfate to tyrosine residues. It takes place in the trans-Golgi, is necessary for the bioactivity of some proteins, and improves their ability to interact with other proteins. In the present work, we show that a protein containing a sulfated tyrosine with a delocalized negative charge forms a salt bridge with another protein if it has two or more adjacent arginine residues containing positive delocalized charges. These noncovalent complexes are so stable that, when submitted to collision induced dissociation, the peptides forming the complex dissociate. Just one covalent bond fragments, the covalent bond between the tyrosine oxygen and the SO3 sulfur, and is represented by the appearance of a new peak (basic peptide + SO3), suggesting that in some instances covalent bonds will break down before the noncovalent bonds between the arginine guanidinium and SO3 dissociate. The data implies that the dissociation pathway is preferred; however, fragmentation between tyrosine and the sulfate residue is a major pathway.
Asunto(s)
Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Sulfatos , Arginina , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Electricidad Estática , Espectrometría de Masas en Tándem , Tirosina/metabolismoRESUMEN
Lipids are major structural components of biomembranes. Negatively charged species such as phosphatidylinositol, phosphatidylserine, sulfatides, and the zwitterionic phosphatidylethanolamines are major components of the cytoplasmic surface of the cellular membrane lipid bilayer and play a key role in several receptors signaling functions. Lipids are not just involved in metabolic and neurological diseases; negatively charged lipids in particular play crucial roles in physiological events such as signal transduction, receptors, and enzymatic activation, as well as storage and release of therapeutic drugs and toxic chemicals in the body. Due to the importance of their role in signaling, the field of lipidomics has rapidly expanded in recent years. In the present study, direct probing of tissue slices with negative ion mode matrix assisted laser desorption/ionization mass spectrometry was employed to profile the distribution of lipids in the brain. In total, 32 lipid species consisting of phosphatidylethanolamines, phosphatidylglycerol, phosphatidylinositols, phosphatidylserines, and sulfatides were assigned. To confirm the structure of lipid species, MALDI-MS/MS analysis was conducted. Product-ion spectra obtained in negative ion mode allow for the assignment of the head groups and the fatty acid chains for the lipid species.