RESUMO
The probe electrospray ionization (PESI) is an ESI-based ionization technique that generates electrospray from the tip of a solid metal needle. In the present work, we describe the PESI mass spectra obtained by in situ measurement of soybeans and several nuts (peanuts, walnuts, cashew nuts, macadamia nuts and almonds) using different solid needles as sampling probes. It was found that PESI-MS is a valuable approach for in situ lipid analysis of these seeds. The phospholipid and triacylglycerol PESI spectra of different nuts and soybean were compared by principal component analysis (PCA). PCA shows significant differences among the data of each family of seeds. Methanolic extracts of nuts and soybean were exposed to air and sunlight for several days. PESI mass spectra were recorded before and after the treatment. Along the aging of the oil (rancidification), the formation of oxidated species with variable number of hydroperoxide groups could be observed in the PESI spectra. The relative intensity of oxidated triacylglycerols signals increased with days of exposition. Monitoring sensitivity of PESI-MS was high. This method provides a fast, simple and sensitive technique for the analysis (detection and characterization) of lipids in seed tissue and degree of oxidation of the oil samples.
Assuntos
Glycine max/química , Juglans/química , Nozes/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Arachis/química , Macadamia/química , Fosfolipídeos/análise , Fosfolipídeos/química , Análise de Componente Principal , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Triglicerídeos/análise , Triglicerídeos/químicaRESUMO
Norharmane is a compound that belongs to a family of alkaloids called ß-carbolines (ßCs). These alkaloids are present in a wide range of biological systems, playing a variety of significant photo-dependent roles. Upon UV-A irradiation, ßCs are able to act as efficient photosensitizers. In this work, we have investigated the photosensitized oxidation of 2'-deoxyadenosine 5'-monophosphate (dAMP) by norharmane in an aqueous phase, upon UV-A (350 nm) irradiation. The effect of the pH was evaluated on both the interactions between norharmane and dAMP in the ground and electronic excited states, and on the dAMP photosensitized oxidation. A quite strong static interaction between norharmane and dAMP was observed, especially under those pH conditions where the protonated form of the alkaloid is present (pH < 7). Theoretical studies were performed to further characterize the static complex structure. The participation of reactive oxygen species (ROS) in the photosensitized reaction was also investigated and the photoproducts were characterized by means of UV-LDI-MS and ESI-MS. All the data provided herein indicate that electron transfer (Type I) within a self-assembled norharmane-dAMP complex is the operative mechanism in the dAMP photosensitization.
Assuntos
Nucleotídeos de Desoxiadenina/química , Harmina/análogos & derivados , Luz , Carbolinas , Harmina/química , Concentração de Íons de Hidrogênio , Estrutura Molecular , Oxirredução , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A group of rhenium (I) complexes including in their structure ligands such as CF(3)SO(3)-, CH(3)CO(2)-, CO, 2,2'-bipyridine, dipyridil[3,2-a:2'3'-c]phenazine, naphthalene-2-carboxylate, anthracene-9-carboxylate, pyrene-1-carboxylate and 1,10-phenanthroline have been studied for the first time by mass spectrometry. The probe electrospray ionization (PESI) is a technique based on electrospray ionization (ESI) that generates electrospray from the tip of a solid metal needle. In this work, mass spectra for organometallic complexes obtained by PESI were compared with those obtained by classical ESI and high flow rate electrospray ionization assisted by corona discharge (HF-ESI-CD), an ideal method to avoid decomposition of the complexes and to induce their oxidation to yield intact molecular cation radicals in gas state [M](+·) and to produce their reduction yielding the gas species [M](-·). It was found that both techniques showed in general the intact molecular ions of the organometallics studied and provided additional structure characteristic diagnostic fragments. As the rhenium complexes studied in the present work showed strong absorption in the UV-visible region, particularly at 355 nm, laser desorption ionization (LDI) mass spectrometry experiments could be conducted. Although intact molecular ions could be detected in a few cases, LDI mass spectra showed diagnostic fragments for characterization of the complexes structure. Furthermore, matrix-assisted laser desorption ionization (MALDI) mass spectra were obtained. Nor-harmane, a compound with basic character, was used as matrix, and the intact molecular ions were detected in two examples, in negative ion mode as the [M](-·) species. Results obtained with 2-[(2E)-3-(4-tert-buthylphenyl)-2-methylprop-2-enylidene] malononitrile (DCTB) as matrix are also described. LDI experiments provided more information about the rhenium complex structures than did the MALDI ones.
Assuntos
Compostos Organometálicos/química , Rênio/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The effect of Ca(2+) (and Mg(2+)) and the disodium salt of ethylenediaminetetraacetic acid (EDTA), a well known Ca(2+) (and Mg(2+)) chelating agent, on the volatilization/ionization of carbohydrates by using electrospray ionization mass spectrometry has been studied. Model compounds such as maltoses (maltose to maltoheptaose), beta-cyclodextrins (beta-cyclodextrin, methyl-beta-cyclodextrin, heptakis(2,6-di-O-methyl)-beta-cyclodextrin, heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin, and 2-hydroxypropyl-beta-cyclodextrin) and fructans (sucrose, 1-ketose, nystose, and 1F-fructofuranosylnystose) were used.
Assuntos
Cálcio/química , Ácido Edético/química , Frutanos/química , Magnésio/química , Maltose/química , Espectrometria de Massas por Ionização por Electrospray/métodos , beta-Ciclodextrinas/químicaRESUMO
The thermal stability of several commonly used crystalline matrix-assisted ultraviolet laser desorption/ionization mass spectrometry (UV-MALDI-MS) matrices, 2,5-dihydroxybenzoic acid (gentisic acid; GA), 2,4,6-trihydroxyacetophenone (THA), alpha-cyano-4-hydroxycinnamic acid (CHC), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid; SA), 9H-pirido[3,4-b]indole (nor-harmane; nor-Ho), 1-methyl-9H-pirido[3,4-b]indole (harmane; Ho), perchlorate of nor-harmanonium ([nor-Ho+H]+) and perchlorate of harmanonium ([Ho+H]+) was studied by heating them at their melting point and characterizing the remaining material by using different MS techniques [electron ionization mass spectrometry (EI-MS), ultraviolet laserdesorption/ionization-time-of-flight-mass spectrometry (UV-LDI-TOF-MS) and electrospray ionization-time-of-flight-mass spectrometry (ESI-TOF-MS)] as well as by thin layer chromatography analysis (TLC), electronic spectroscopy (UV-absorption, fluorescence emission and excitation spectroscopy) and 1H nuclear magnetic resonance spectroscopy (1H-NMR). In general, all compounds, except for CHC and SA, remained unchanged after fusion. CHC showed loss of CO2, yielding the trans-/cis-4-hydroxyphenylacrilonitrile mixture. This mixture was unambiguously characterized by MS and 1H-NMR spectroscopy, and its sublimation capability was demonstrated. These results explain the well-known cluster formation, fading (vanishing) and further recovering of CHC when used as a matrix in UV-MALDI-MS. Commercial SA (SA 98%; trans-SA/cis-SA 5:1) showed mainly cis- to-trans thermal isomerization and, with very poor yield, loss of CO2, yielding (3',5'-dimethoxy-4'-hydroxyphenyl)-1-ethene as the decarboxilated product. These thermal conversions would not drastically affect its behavior as a UV-MALDI matrix as happens in the case of CHC. Complementary studies of the photochemical stability of these matrices in solid state were also conducted.
Assuntos
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Acetofenonas/química , Carbolinas , Ácidos Cumáricos/química , Gentisatos/química , Harmina/análogos & derivados , Harmina/química , Temperatura Alta , Espectroscopia de Ressonância Magnética , Transição de Fase , Fotoquímica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentaçãoRESUMO
Sulfated glycosphingolipids are present on the surface of a variety of cells. They are active participants in adhesion processes in many systems and appear to be involved in the regulation of cell proliferation, differentiation and other developmental cellular events. However, the body of knowledge about synthesis, structure, and function of glycolipids in parasitic protozoa is very limited so far. In this work, we show by metabolic incorporation of [(14)C]palmitic acid, [(14)C]glucose and Na(2)(35)SO(4) that sulfoglycosphingolipids are biosynthesized in the three intraerythrocytic stages of Plasmodium falciparum. After saponification, purification of the labelled acidic components was achieved and two components named SPf1 and SPf2 were characterized. Chemical degradations and TLC analysis pointed out to sulfolipidic structures. Analysis by UV-MALDI-TOF mass spectrometry in the negative ion mode using nor-harmane as matrix showed for SPf2 a structure consisting in a disulfated hexose linked to a 20:1 sphingosine acylated with C18:0 fatty acid. Interestingly, parasite treatment with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) caused an arrest on parasite development associated to the inhibition of sulfoglycolipid biosynthesis. Taking into account that sulfoglycolipidic structures are currently involved in adhesion processes, our findings open the possibility to study the participation of this type of structures in the described aggregation phenomena in severe malaria and may contribute to clarify the pathogenesis of the disease. This report shows for the first time the synthesis of sulfoglycolipids in Apicomplexa.
Assuntos
Plasmodium falciparum/metabolismo , Sulfoglicoesfingolipídeos/metabolismo , Animais , Antimaláricos/farmacologia , Radioisótopos de Carbono/metabolismo , Cromatografia em Camada Fina , Glucose/metabolismo , Espectrometria de Massas , Morfolinas/farmacologia , Ácido Palmítico/metabolismo , Plasmodium falciparum/química , Plasmodium falciparum/efeitos dos fármacos , Esfingolipídeos/farmacologia , Sulfatos/metabolismo , Sulfoglicoesfingolipídeos/análiseRESUMO
The isolation, purification and analysis of the lipid A obtained from Mesorhizobium loti Ayac 1 BII strain is presented. Analysis of the carbohydrate moiety after acid hydrolysis by high-pH anion-exchange chromatography with pulse amperometric detection (HPAEC-PAD) showed the presence of glucosamine and galacturonic acid as the only sugar components. Gas chromatographic (GC) and GC/mass spectrometric (MS) analysis of the fatty acids revealed the presence of 3-OH-C12:0; 3-OH-C13:0; 3-OH-C20:0 and 27-OH-C28:0 among the major hydroxylated species. In addition, C16:0, C17:0, C18:0 and C 20:0 were shown as main saturated fatty acids. Different polyacylated species were evidenced by thin layer chromatography of lipid A, allowing the purification of two fractions. Ultraviolet matrix-assisted laser desorption/ionization time-of-flight (UV-MALDI-TOF) MS analysis with different matrices, in the positive- and negative-ion mode, was performed. The fast moving component revealed the presence of hexa-acylated species, varying in the fatty acid composition. Species containing three 3-OH fatty acids and a 27-OH-C28:0 fatty acid were observed. Individual ions within this family differ by +/-14 mass units. The slow moving component was enriched mainly in penta-acylated species. Among them, three subgroups were detected: the major one compatible with the sugar core bearing two 3-OH 20:0 fatty acids, a 3-OH 13:0 or a 3-OH 12:0 fatty acid, a 27-OH 28:0 fatty acid and one saturated fatty acid. Each signal differs in a C18:0 acyl unit from the corresponding hexa-acylated family. On the other hand, a subgroup bearing one 3-OH 20:0 fatty acid, one 27-OH 28:0 fatty acid and two non-polar fatty acids was shown. A minor subgroup compatible with structures containing two hydroxylated and three non-polar fatty acids was also detected. The results obtained showed that nor-harmane was an excellent matrix for charged lipid A structural studies in both, positive and negative ion modes.
Assuntos
Lipídeo A/química , Proteobactérias/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cromatografia em Camada Fina/métodos , Ácidos Graxos/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glucosamina/análise , Ácidos Hexurônicos/análise , Lipídeo A/análiseRESUMO
Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine proteinase, cruzipain. This lysosomal enzyme bears an unusual C-terminal extension that contains a number of post-translational modifications, and most antibodies in natural and experimental infections are directed against it. In this report we took advantage of UV-MALDI-TOF mass spectrometry in conjunction with peptide N-glycosidase F deglycosylation and high performance anion exchange chromatography analysis to address the structure of the N-linked oligosaccharides present in this domain. The UV-MALDI-TOF MS analysis in the negative-ion mode, using nor-harmane as matrix, allowed us to determine a new striking feature in cruzipain: sulfated high-mannose type oligosaccharides. Sulfated GlcNAc2Man3 to GlcNAc2Man9 species were identified. In accordance, after chemical or enzymatic desulfation, the corresponding signals disappeared. In addition, by UV-MALDI-TOF MS analysis (a) a main population of high-mannose type oligosaccharides was shown in the positive-ion mode, (b) lactosaminic glycans were also identified, among them, structures corresponding to monosialylated species were detected, and (c) as an interesting fact a fucosylated oligosaccharide was also detected. The presence of the deoxy sugar was further confirmed by high performance anion exchange chromatography. In conclusion, the total number of oligosaccharides occurring in cruzipain was shown to be much higher than previous estimates. This constitutes the first report on the presence of sulfated glycoproteins in Trypanosomatids.
Assuntos
Cisteína Endopeptidases/química , Oligossacarídeos de Cadeias Ramificadas/química , Trypanosoma cruzi/enzimologia , Animais , Cisteína Endopeptidases/metabolismo , Eletroforese em Gel de Poliacrilamida , Fucose/química , Fucose/metabolismo , Manose/química , Manose/metabolismo , Oligossacarídeos de Cadeias Ramificadas/metabolismo , Proteínas de Protozoários , Coloração pela Prata , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de TempoRESUMO
Malaria remains a major health problem especially in tropical and subtropical regions of the world, and therefore developing new antimalarial drugs constitutes an urgent challenge. Lipid metabolism has been attracting a lot of attention as an application for malarial chemotherapeutic purposes in recent years. However, little is known about glycosphingolipid biosynthesis in Plasmodium falciparum. In this report we describe for the first time the presence of an active glucosylceramide synthase in the intraerythrocytic stages of the parasite. Two different experiments, using UDP-[(14)C]glucose as donor with ceramides as acceptors, or UDP-glucose as donor and fluorescent ceramides as acceptors, were performed. In both cases, we found that the parasitic enzyme was able to glycosylate only dihydroceramide. The enzyme activity could be inhibited in vitro with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP). In addition, de novo biosynthesis of glycosphingolipids was shown by metabolic incorporation of [(14)C]palmitic acid and [(14)C]glucose in the three intraerythrocytic stages of the parasite. The structure of the ceramide, monohexosylceramide, trihexosylceramide and tetrahexosylceramide fractions was analysed by UV-MALDI-TOF mass spectrometry. When PPMP was added to parasite cultures, a correlation between arrest of parasite growth and inhibition of glycosphingolipid biosynthesis was observed. The particular substrate specificity of the malarial glucosylceramide synthase must be added to the already known unique and amazing features of P. falciparum lipid metabolism; therefore this enzyme might represent a new attractive target for malarial chemotherapy.