Electrospray ionization mass spectrometry analysis of changes in phospholipids in RBL-2H3 mastocytoma cells during degranulation

Biological membranes contain an extraordinary diversity of lipids. Phospholipids function as major structural elements of cellular membranes, and analysis of changes in the highly heterogeneous mixtures of lipids found in eukaryotic cells is central to understanding the complex functions in which lipids participate. Phospholipase-catalyzed hydrolysis of phospholipids often follows cell surface receptor activation. Recently, we demonstrated that granule fusion is initiated by addition of exogenous, nonmammalian phospholipases to permeabilized mast cells. To pursue this finding, we use positive and negative mode Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to measure changes in the glycerophospholipid composition of total lipid extracts of intact and permeabilized RBL-2H3 (mucosal mast cell line) cells. The low energy of the electrospray ionization results in efficient production of molecular ions of phospholipids uncomplicated by further fragmentation, and changes were observed that eluded conventional detection methods. From these analyses we have spectrally resolved more than 130 glycerophospholipids and determined changes initiated by introduction of exogenous phospholipase C, phospholipase D, or phospholipase A2. These exogenous phospholipases have a preference for phosphatidylcholine with long polyunsaturated alkyl chains as substrates and, when added to permeabilized mast cells, produce multiple species of mono- and polyunsaturated diacylglycerols, phosphatidic acids, and lysophosphatidylcholines, respectively. The patterns of changes of these lipids provide an extraordinarily rich source of data for evaluating the effects of specific lipid species generated during cellular processes, such as exocytosis.

Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.

The binding stoichiometry of gene V protein from bacteriophage f1 to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution. Addition of oligonucleotides resulted in formation of protein-oligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry. The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of protein-oligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful too] in the study of protein-DNA interactions.

Avaliação de técnicas cromatográficas acopladas a espectrometria de massas para análise de morfolina em manga

A constante preocupação com o aumento do uso de agrotóxicos nas lavouras e os riscos gerados pelos resíduos destes compostos fazem com que os órgãos responsáveis pela fiscalização de alimentos no Brasil controlem a presença dessas substâncias nos produtos que chegam à mesa do consumidor. Atualmente, um dos grandes problemas na produção de alimentos é a utilização de substâncias proibidas em lavouras, muitas das quais não possuem estudos nem limites máximos de resíduos (LMR) estabelecidos, assim como a utilização de substâncias já registradas, mas em quantidades ou métodos de manejo incorretos. Ambos os casos podem resultar em sérios problemas à saúde humana. O objetivo deste estudo foi a avaliação da determinação de morfolina em amostras de manga utilizando técnicas como a Extração em Fase Sólida e a Cromatografia Gasosa acoplada à Espectrometria de Massas (SPE-GC-MS), assim como a Microextração em Sorvente Empacotado e Cromatografia Gasosa acoplada à Espectrometria de Massas (MEPS-GC-MS). Um segundo objetivo deste estudo consistiu em desenvolver, validar e avaliar uma metodologia analítica capaz de identificar quantitativamente a morfolina em amostras de manga por Cromatografia Líquida de Ultra Eficiência acoplada a Espectrometria de Massas em tandem (UHPLC-MS/MS). Para análise por GC-MS fez-se necessária a etapa de derivatização do analito, de forma que o mesmo aumentasse sua volatilidade e diminuísse a polaridade. A comparação entre as técnicas SPE e MEPS não foi possível devido ao efeito de matriz causado pela contaminação do liner e da coluna cromatográfica. Já a metodologia validada por UHPLC-MS/MS seguiu os critérios exigidos pelo Manual de Garantia da Qualidade Analítica, do Ministério da Agricultura Pecuária e Abastecimento (MAPA). O método foi aplicado em mangas de diferentes variedades obtidas no comércio local. Não foram encontrados resíduos de morfolina em nenhuma das amostras investigadas, de acordo com a metodologia proposta. Os resultados apresentados neste trabalho estabelecem metodologias eficientes, rápidas e de baixo custo na determinação de morfolina em amostras de manga.

Extração, determinação da composição fenólica e avaliação do potencial de desativação de espécies reativas de oxigênio e da atividade anti-inflamatória de resíduos de amendoim, pimenta-rosa e pimenta-do-reino

O setor agroindustrial tem se expandido muito nos últimos anos, levando o país a um aumento na geração de resíduos agroindustriais, sendo que a maior parte deles ainda é descartada no meio ambiente, sem tratamento adequado, ou utilizada na alimentação animal, destinos que a priori, não geram ganhos econômicos para a agroindústria além de representarem gargalos logísticos e ambientais na sua disposição. Nesse sentido, o presente trabalho teve como objetivos otimizar o processo de extração de compostos bioativos, avaliar in vitro as atividades antioxidante, por meio da desativação de espécies reativas de oxigênio, e anti-inflamatória, bem como determinar a composição fenólica dos resíduos agroindustriais a saber: película de amendoim (Arachis hypogaea) (cultivares IAC886 e IAC505), pimenta-rosa (Schinus terebinthifolius Raddi) e pimenta-do-reino (Piper Nigrum L). O processo de otimização da extração de compostos antioxidantes foi realizado utilizando dois processos de extração, extração convencional e subcrítica, em delineamento composto central rotacional, utilizando como variáveis a temperatura e tempo e a pressão e temperatura, respectivamente, com os solventes etanol 80%, água e propilenoglicol 80%. Durante o processo de otimização a atividade antioxidante foi avaliada pelo método de sequestro do radical ABTS. Os melhores resultados foram obtidos para a extração convencional com os solventes etanol 80%, água e propilenoglicol 80%. A película de amendoim (IAC505) apresentou as maiores atividades antioxidantes (1.396,67, 580,44 e 859,89 μmol.g-1 em equivalentes de trolox, para os solventes etanol 80%, água e propilenoglicol 80%, respectivamente). A partir dos resultados obtidos para os solventes testados, utilizando a extração convencional, foram feitas outras análises de atividade antioxidante considerando o tempo e temperatura ideal de extração. Foram realizadas análises de avaliação da capacidade de redução de Folin-Ciocalteau e potencial de desativação dos radicais livres sintéticos (DPPH e ABTS) e espécies reativas de oxigênio (radicais peroxila, superóxido e ácido hipocloroso). O solvente de extração que apresentou melhores resultados em todos os ensaios foi o etanol 80%, sendo, portanto o solvente utilizado nas análises subsequentes. A partir da definição do melhor sistema extrator foram realizadas análises da composição fenólica, por meio das técnicas de cromatografia líquida de alta eficiência em modo analítico (HPLC-RP), cromatografia gasosa acoplada com espectrometria de massas (GC-MS), e avaliação in vitro da atividade anti-inflamatória. Foram identificados nos resíduos estudados procianidinas B1 e B2, ácido p-cumárico, miricetina, ácido ferúlico, ácido siríngico, ácido sinápico, epicatequina e catequina. A pimenta-do-reino diminui significativamente os níveis de TNF-α e nitritos, reduzindo assim o processo inflamatório gerado. Os resultados obtidos neste trabalho demonstram que estes resíduos agroindústrias possuem grande potencial biológico, podendo assim ser melhores aproveitados tanto pela indústria de alimentos quanto pela indústria farmacêutica.

Disintegrability under composting conditions of plasticized PLA–PHB blends

The disintegration under composting conditions of films based on poly(lactic acid)–poly(hydroxybutyrate) (PLA–PHB) blends and intended for food packaging was studied. Two different plasticizers, poly(ethylene glycol) (PEG) and acetyl-tri-n-butyl citrate (ATBC), were used to limit the inherent brittleness of both biopolymers. Neat PLA, plasticized PLA and PLA–PHB films were processed by melt-blending and compression molding and they were further treated under composting conditions in a laboratory-scale test at 58 ± 2 °C. Disintegration levels were evaluated by monitoring their weight loss at different times: 0, 7, 14, 21 and 28 days. Morphological changes in all formulations were followed by optical and scanning electron microscopy (SEM). The influence of plasticizers on the disintegration of PLA and PLA–PHB blends was studied by evaluating their thermal and nanomechanical properties by thermogravimetric analysis (TGA) and the nanoindentation technique, respectively. Meanwhile, structural changes were followed by Fourier transformed infrared spectroscopy (FTIR). The ability of PHB to act as nucleating agent in PLA–PHB blends slowed down the PLA disintegration, while plasticizers speeded it up. The relationship between the mesolactide to lactide forms of PLA was calculated with a Pyrolysis–Gas Chromatography–Mass Spectrometry device (Py–GC/MS), revealing that the mesolactide form increased during composting.

Non-spectral interferences due to the presence of sulfuric acid in inductively coupled plasma mass spectrometry

Results of a systematic study concerning non-spectral interferences from sulfuric acid containing matrices on a large number of elements in inductively coupled plasma–mass spectrometry (ICP-MS) are presented in this work. The signals obtained with sulfuric acid solutions of different concentrations (up to 5% w w− 1) have been compared with the corresponding signals for a 1% w w− 1− nitric acid solution at different experimental conditions (i.e., sample uptake rates, nebulizer gas flows and r.f. powers). The signals observed for 128Te+, 78Se+ and 75As+ were significantly higher when using sulfuric acid matrices (up to 2.2-fold for 128Te+ and 78Se+ and 1.8-fold for 75As+ in the presence of 5 w w-1 sulfuric acid) for the whole range of experimental conditions tested. This is in agreement with previously reported observations. The signal for 31P+ is also higher (1.1-fold) in the presence of sulfuric acid. The signal enhancements for 128Te+, 78Se+, 75As+ and 31P+ are explained in relation to an increase in the analyte ion population as a result of charge transfer reactions involving S+ species in the plasma. Theoretical data suggest that Os, Sb, Pt, Ir, Zn and Hg could also be involved in sulfur-based charge transfer reactions, but no experimental evidence has been found. The presence of sulfuric acid gives rise to lower ion signals (about 10–20% lower) for the other nuclides tested, thus indicating the negative matrix effect caused by changes in the amount of analyte loading of the plasma. The elemental composition of a certified low-density polyethylene sample (ERM-EC681K) was determined by ICP-MS after two different sample digestion procedures, one of them including sulfuric acid. Element concentrations were in agreement with the certified values, irrespective of the acids used for the digestion. These results demonstrate that the use of matrix-matched standards allows the accurate determination of the tested elements in a sulfuric acid matrix.

Maize Stress DIESI-MS Data and R Script

Climatic change is an increasing challenge for agriculture that is driving the development of suitable crops in order to ensure supply for both human nutrition and animal feed. In this context, it is increasingly important to understand the biochemical responses of cells to environmental cues at the whole system level, an aim that is being brought closer by advances in high throughput, cost-efficient plant metabolomics. To support molecular breeding activities, we have assessed the economic, technical and statistical feasibility of using direct mass spectrometry methods to evaluate the physiological state of maize (Zea mays L.) plants grown under different stress conditions.

Organic matter based proxies from surface sediments along three transects on the Pakistan continental margin

A valid assessment of selective aerobic degradation on organic matter (OM) and its impact on OM-based proxies is vital to produce accurate environmental reconstructions. However, most studies investigating these effects suffer from inherent environmental heterogeneities. In this study, we used surface samples collected along two meter-scale transects and one longer transect in the northeastern Arabian Sea to constrain initial OM heterogeneity, in order to evaluate selective aerobic degradation on temperature, productivity and alteration indices at the sediment-water interface. All of the studied alteration indices, the higher plant alkane index, alcohol preservation index, and diol oxidation index, demonstrated that they are sensitive indicators for changes in the oxygen regime. Several export production indices, a cholesterol-based stanol/stenol index and dinoflagellate lipid- and cyst-based ratios, showed significant (more than 20%) change only over the lateral oxygen gradients. Therefore, these compounds do not exclusively reflect surface water productivity, but are significantly altered after deposition. Two of the proxies, glycerol dibiphytanyl glycerol tetraether-based TEX86 sea surface temperature indices and indices based on phytol, phytane and pristane, did not show any trends related to oxygen. Nevertheless, unrealistic sea surface temperatures were obtained after application of the TEX86, TEX86L, and TEX86H proxies. The phytol-based ratios were likely affected by the sedimentary production of pristane. Our results demonstrate the selective impact of aerobic organic matter degradation on the lipid and palynomorph composition of surface sediments along a short lateral oxygen gradient and suggest that some of the investigated proxies may be useful tracers of changing redox conditions at the sediment-water interface.

Chemical and functional identification and characterization of novel sulfated alpha-conotoxins from the cone snail Conus anemone

An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the -conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3/beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr(16)]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7(IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.

A rapid HPLC-mass spectrometry cyclosporin method suitable for current monitoring practices

Objectives: Cyclosporin is an immunosuppressant drug with a narrow therapeutic window. Trough and 2-h post-dose blood samples are currently used for therapeutic drug monitoring in solid organ transplant recipients. The aim of the current study was to develop a rapid HPLC-tandem mass spectrometry (HPLC-MS) method for the measurement of cyclosporin in whole blood that was not only suitable for the clinical setting but also considered a reference method. Methods: Blood samples (50 mu L) were prepared by protein precipitation followed by C-18 solid-phase extraction while using d(12) cyclosporin as the internal standard. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface in positive ionization mode. Results: The assay was linear from 10 to 2000 mu g/L (r(2) > 0.996, n = 9). Inter-day,analytical recovery and imprecision using whole blood quality control samples at 10, 30, 400, 1500, and 2000 mu g/L were 94.9-103.5% and

Uptake and metabolism of ginsenoside Rh2 and its aglycon protopanaxadiol by Caco-2 cells

The uptake and metabolism profiles of ginsenoside Rh2 and its aglycon protopanaxadiol (ppd) were studied in the human epithelial Caco-2 cell line. High-performance liquid chromatography-mass spectrometry was applied to determine Rh2 and its aglycon ppd concentration in the cells at different pH, temperature, concentration levels and in the presence or absence of inhibitors. Rh2 uptake was time and concentration dependent, and its uptake rates were reduced by metabolic inhibitors and influenced by low temperature, thus indicating that the absorption process was energy-dependent. Drug uptake was maximal when the extracellular pH was 7.0 for Rh2 and 8.0 for ppd. Rh2 kinetic analysis showed that a non-saturable component (K-d 0.17 nmol (.) h(-1) (.) mg(-1) protein) and an active transport system with a K-m of 3.95 mumol (.) l(-1) and a V-max of 4.78 nmol(.)h(-1) (.)mg(-1) protein were responsible for the drug uptake. Kinetic analysis of ppd showed a non-saturable component (K-d 0.78 nmol (.) h(-1) (.) mg(-1) protein). It was suggested that active extrusion of P-glycoprotein and drug degradation in the intestine may influence Rh2 bioavailability.

ICP-MS Trace Element Analysis of Song Dynasty Porcelains from Ding, Jiexiu and Guantai kilns, North China

Bodies of Ding kiln white porcelains and their imitations from Guantai and Jiexiu kilns of the Chinese Song dynasty (960-1279 AD) were analysed for 40 trace elements by inductively coupled plasma mass spectrometry (ICP-MS). Numerous trace element compositions and ratios allow these visually similar products to be distinguished, and a Ding-style shard of uncertain origin is identified as a likely genuine Ding product. In Jiexiu kiln, Ding-style products have trace element features distinctive from blackwares of an inferior quality intended for the lower end market. Based on geochemical behaviour of these trace elements, we propose that geochemically distinctive raw materials were used for Ding-style products of a higher quality, which possibly also underwent purification by levigation prior to use. Capable of analysing over 40 elements with a typical long term precision of < 2%, this high precision ICP-MS method proves to be very powerful for grouping and characterising archaeological ceramics. Combined with geochemical interpretation, it can provide insights into the raw materials and techniques used by ancient potters. (C) 2004 Elsevier Ltd. All rights reserved.

Folate: Methods of analysis

Folates and its derivatives occur as polyglutamates in nature. The multiplicity of forms and the generally low levels in foods makes quantitative analysis of folate a difficult task. The assay of folates from foods generally involves three steps: liberation of folates from the cellular matrix; deconjugation from the polyglutamate to the mono and di-glutamate forms; and the detection of the biological activity or chemical concentration of the resulting folates. The detection methods used are the microbiological assay relying on the turbidimetric bacterial growth of Lactobacillus rhamnosus which is by far the most commonly used method; the HPLC and LC/MS techniques and bio-specific procedures. This review attempts to describe the methods along with the merits and demerits of using each of these methods.

Investigation of healthy and infected newborn saliva: the role of proteins and lipids as potential inflammatory diagnostic tools

We have carried out a discovery proteomics investigation aimed at identifying disease biomarkers present in saliva, and, more specifically, early biomarkers of inflammation. The proteomic characterization of saliva is possible due to the straightforward and non-invasive sample collection that allows repetitive analyses for pharmacokinetic studies. These advantages are particularly relevant in the case of newborn patients. The study was carried out with samples collected during the first 48 hours of life of the newborns according to an approved Ethic Committee procedure. In particular, the salivary samples were collected from healthy and infected (n=1) newborns. Proteins were extracted through cycles of sonication, precipitated in ice cold acetone, resuspended and resolved by 2D-electrophoresis. MALDI TOF/TOF mass spectrometry analysis was performed for each spot obtaining the proteins’ identifications. Then we compared healthy newborn salivary proteome and an infected newborn salivary proteome in order to investigate proteins differently expressed in inflammatory condition. In particular the protein alpha-1-antitrypsin (A1AT), correlated with inflammation, was detected differently expressed in the infected newborn saliva. Therefore, in the second part of the project we aimed to develop a robust LC-MS based method that identifies and quantifies this inflammatory protein within saliva that might represent the first relevant step to diagnose a condition of inflammation with a no-invasive assay. The same LC-MS method is also useful to investigate the presence of the F allelic variant of the A1AT in biological samples, which is correlated with the onset of pulmonary diseases. In the last part of the work we analysed newborn saliva samples in order to investigate how phospholipids and mediators of inflammation (eicosanoids) are subject to variations under inflammatory conditions and a trend was observed in lysophosphatidylcholines composition according to the inflammatory conditions.

Proteomic analysis of a noninvasive human model of acute inflammation and its resolution:the twenty-one day gingivitis model

The 21-day experimental gingivitis model, an established noninvasive model of inflammation in response to increasing bacterial accumulation in humans, is designed to enable the study of both the induction and resolution of inflammation. Here, we have analyzed gingival crevicular fluid, an oral fluid comprising a serum transudate and tissue exudates, by LC-MS/MS using Fourier transform ion cyclotron resonance mass spectrometry and iTRAQ isobaric mass tags, to establish meta-proteomic profiles of inflammation-induced changes in proteins in healthy young volunteers. Across the course of experimentally induced gingivitis, we identified 16 bacterial and 186 human proteins. Although abundances of the bacterial proteins identified did not vary temporally, Fusobacterium outer membrane proteins were detected. Fusobacterium species have previously been associated with periodontal health or disease. The human proteins identified spanned a wide range of compartments (both extracellular and intracellular) and functions, including serum proteins, proteins displaying antibacterial properties, and proteins with functions associated with cellular transcription, DNA binding, the cytoskeleton, cell adhesion, and cilia. PolySNAP3 clustering software was used in a multilayered analytical approach. Clusters of proteins that associated with changes to the clinical parameters included neuronal and synapse associated proteins.