Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INFalpha and INFgamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INF alpha and INF gamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

Pinic and pinonic acid formation in the reaction of ozone with alpha-pinene

The mechanism of formation of key compounds in atmospheric secondary aerosol (SOA) has been investigated by studying the products of the ozonolysis of an enal derived from alpha-pinene using gas chromatography coupled to mass spectrometry.

Chromatographic alignment of LC-MS and LC-MS/MS datasets by genetic algorithm feature extraction

Liquid chromatography-mass spectrometry (LC-MS) datasets can be compared or combined following chromatographic alignment. Here we describe a simple solution to the specific problem of aligning one LC-MS dataset and one LC-MS/MS dataset, acquired on separate instruments from an enzymatic digest of a protein mixture, using feature extraction and a genetic algorithm. First, the LC-MS dataset is searched within a few ppm of the calculated theoretical masses of peptides confidently identified by LC-MS/MS. A piecewise linear function is then fitted to these matched peptides using a genetic algorithm with a fitness function that is insensitive to incorrect matches but sufficiently flexible to adapt to the discrete shifts common when comparing LC datasets. We demonstrate the utility of this method by aligning ion trap LC-MS/MS data with accurate LC-MS data from an FTICR mass spectrometer and show how hybrid datasets can improve peptide and protein identification by combining the speed of the ion trap with the mass accuracy of the FTICR, similar to using a hybrid ion trap-FTICR instrument. We also show that the high resolving power of FTICR can improve precision and linear dynamic range in quantitative proteomics. The alignment software, msalign, is freely available as open source.

In vitro fermentation by human fecal microflora of wheat arabinoxylans

The fermentation of three arabinoxylan (AX) fractions from wheat by the human fecal microflora was investigated in vitro. Three AX fractions, with average molecular masses of 354, 278, and 66 kDa, were incorporated into miniature-scale batch cultures (with inulin as a positive prebiotic control) with feces from three healthy donors, aged 23-29. Microflora changes were monitored by the culture-independent technique, fluorescent in situ hybridization, and short chain fatty acid (SCFA) and lactic acid production were measured by high-performance liquid chromatography. Total cell numbers increased significantly in all treated cultures, and the fermentation of AX was associated with a proliferation of the bifidobacteria, lactobacilli, and eubacteria groups. Smaller but statistically significant increases in bacteroides and clostridia groups were also observed. All AX fractions had comparable bifidogenic impacts on the microflora at 5 and 12 h, but the 66 kDa AX was particularly selective for lactobacilli. Eubacteria increased significantly on all AX fractions, particularly on 66 kDa AX. As previously reported, inulin gave a selective increase in bifidobacteria. All supplemented cultures showed significant rises in total SCFA production, with a particularly high proportion of butyric acid being produced from AX fermentation. The prebiotic effect, that is, the selectivity of AX for bifidobacteria and lactobacilli groups, increased as the molecular mass of the AX decreased. This suggests that molecular mass may influence the fermentation of AX in the colon.

Comparison of bottom-up protein identification methods

With the rapid development of proteomics, a number of different methods appeared for the basic task of protein identification. We made a simple comparison between a common liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow using an ion trap mass spectrometer and a combined LC-MS and LC-MS/MS method using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and accurate peptide masses. To compare the two methods for protein identification, we grew and extracted proteins from E. coli using established protocols. Cystines were reduced and alkylated, and proteins digested by trypsin. The resulting peptide mixtures were separated by reversed-phase liquid chromatography using a 4 h gradient from 0 to 50% acetonitrile over a C18 reversed-phase column. The LC separation was coupled on-line to either a Bruker Esquire HCT ion trap or a Bruker 7 tesla APEX-Qe Qh-FTICR hybrid mass spectrometer. Data-dependent Qh-FTICR-MS/MS spectra were acquired using the quadrupole mass filter and collisionally induced dissociation into the external hexapole trap. Proteins were in both schemes identified by Mascot MS/MS ion searches and the peptides identified from these proteins in the FTICR MS/MS data were used for automatic internal calibration of the FTICR-MS data, together with ambient polydimethylcyclosiloxane ions.

Characterisation of secondary metabolites associated with neutrophil apoptosis

We studied changes in secondary metabolites in human neutrophils undergoing constitutive or tumour necrosis factor (TNFalpha) stimulated apoptosis by a combination of high-performance liquid chromatography (HPLC) and NMR spectroscopy. Our results show that in contrast to freshly isolated neutrophils, neutrophil cells aged for 20 h in vitro had marked differences in the levels of a number of endogenous metabolites including lactate, amino acids and phosphocholine (PCho). There was no change in the concentration of taurine or glutamate and the ATP/ADP ratio was not affected. Levels of glutamine and lactate actually decreased. Identical changes were also observed in neutrophils stimulated to undergo apoptosis over a shorter time period (6 h) in the presence of TNFalpha and the phosphatidylinositol-3-kinase inhibitor wortmannin (WM). The changes in the concentration of PCho suggest possible activation of phospholipase associated with apoptosis or a selective failure of phosphatidycholine synthesis. The increased levels of apoptosis obtained with WM+TNFalpha, compared to TNFalpha by itself, suggest a synergistic effect by these compounds. The acceleration in rate of apoptosis probably arises from suppression by WM of pathway(s) that normally delay the onset of apoptosis. Changes in PCho and other endogenous metabolites, if proven to be characteristic of apoptosis in other cell systems, may permit non-invasive quantification of apoptosis. '

Brazilian red propolis: unreported substances, antioxidant and antimicrobial activities

BACKGROUND: Chloroform, ethyl acetate and methanol extracts of a sample of red propolis from the state of Alagoas (northeast Brazil) were analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry. Antimicrobial and antioxidant activities were also obtained. RESULTS: The propolis sample contained low content of narigenin-8-C-hexoside, this being the first report of a C-glycoside in propolis. The main constituent found was characterized as 3,4,2`,3`-tetrahydroxychalcone. Other important constituents were the chalcone isoliquiritigenin, the isoflavans (3S)-vestitol, (3S)-7-O-methylvestitol, the pterocarpan medicarpin, the phenylpropenes trans-anethol, methyl eugenol, elimicin, methoxyeugenol and cis-asarone, and the triterpenic alcohols lupeol and alpha- and beta- amyrins. The methanol extract exhibited high antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl and beta-carotene/linoleic acid assay methods, and antimicrobial activity toward Gram-positive and Gram-negative bacteria. CONCLUSION: Structures are suggested for new substances never before seen in any kind of propolis. This is the first report of 3,4,2`,3`-tetrahydroxychalcone and a flavone C-glycoside in a propolis sample. (C) 2011 Society of Chemical Industry

Leishmania amazonensis: Biosynthesis of polyprenols of 9 isoprene units by amastigotes

The isoprenoid metabolic pathway in protozoa of the Leishmania genus exhibits distinctive characteristics. These parasites, as well as other members of the Trypanosomatidae family, synthesize ergosterol, instead of cholesterol, as the main membrane sterol lipid. Leishmania has been shown to utilize leucine, instead of acetate as the main precursor for sterol biosynthesis. While mammalian dolichols are molecules containing 15-23 isoprene units, Leishmania amazonensis promastigotes synthesize dolichol of 11 and 12 units. In this paper, we show that the intracellular stages of L. amazonensis, amastigotes, synthesize mainly polyprenols of 9 isoprene units, instead of dolichol. (c) 2007 Elsevier Inc. All rights reserved.

A powder X-ray diffraction method for detection of polyprenylated benzophenones in plant extracts associated with HPLC for quantitative analysis

A robust, direct, rapid and non-destructive X-ray diffraction crystallography method to detect the polyprenylated benzophenones 7-epi-clusianone (1) and guttiferone A (2) in extracts from Garcinia brasiliensis is presented. Powder samples of benzophenones 1 and 2, dried hexane extracts from G. brasiliensis seeds and fruit`s pericarp, and the dried ethanolic extract from G. brasiliensis seeds were unambiguously characterized by powder X-ray diffractometry. The calculated X-ray diffraction peaks from crystal structures of analytes 1 and 2, previously determined by single-crystal X-ray diffraction technique, were overlaid to those of the experimental powder diffractograms, providing a practical identification of these compounds in the analyzed material and confirming the pure contents of the powder samples. Using the X-ray diffraction crystallography method, the studied polyprenylated benzophenones were selectively and simultaneously detected in the extracts which were mounted directly on sample holder. In addition, reference materials of the analytes were not required for analyses since the crystal structures of the compounds are known. High performance liquid chromatography analyses also were comparatively carried out to quantify the analytes in the same plant extracts showing to be in agreement with X-ray diffraction crystallography method. (C) 2010 Elsevier B.V. All rights reserved.

Comparative bioavailability of three ibuprofen formulations in healthy human volunteers

Objective: To assess the bioequivalence of three ibuprofen formulations (Test formulation: ibuprofen (400 mg capsule) manufactured by Cardinal Health Brasil 402 Ltda. (Sorocaba, Brazil) and licensed to Boehringer Ingelheim do Brasil Quim. e Farm. Ltda. (Sao Paulo, Brazil); Reference formulation (1): ibuprofen (Advil (R); 2 x 200 mg coated tablet) from Wyeth-Whitehall Ltda. (Itapevi, Brazil); Reference formulation (2): ibuprofen (Alivium (R); 8 ml x 50 mg/ml solution) from Schering Plough S.A. (Rio de Janeiro, Brazil)) in 24 healthy volunteers of both sexes. Methods: The study was conducted using an open, randomized, three-period crossover design with at least 5-day washout interval. Plasma samples were obtained over a 24-h period. Plasma ibuprofen concentrations were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with negative ion electrospray ionization using multiple reaction monitoring (MRM). The following pharmacokinetic parameters were obtained from the ibuprofen plasma concentration vs. time curves: AUC(last), AUC(trunctmax) AUC(inf) and C-max. Results: The limit of quantification for ibuprofen was 0.1 mu g x ml(-1). The geometric mean with corresponding 90% confidence interval (CI) for Test/Reference (1) percent ratios were 114.24% (90% CI = 105.67, 123.50%) for C-max, 98.97% (90% CI = 94.69, 103.44%) for AUC(last) and 99.40% (90% CI = 95.21, 103.78%) for AUCinf. The geometric mean and respective 90% confidence interval (CI) for Test/Reference (2) percent ratios were 108.38% (90% Cl = 100.195, 117.25%) for C-max, 100.79% (90% CI = 96.39, 105.40%) for AUC(last) and 101.26% (90% CI = 96.94, 105.77%) for AUC(inf); t(max) for the 400 mg Test capsule was shorter than that for the 2 x 200 mg Reference (1) tablets (p < 0.002). Conclusion: Since the 90% CI for AUC(last), AUC(inf) and C-max ratios were within the 80 - 125% interval proposed by the US FDA, it was concluded that ibuprofen formulation manufactured by Cardinal Health Brasil 402 Ltda. and licensed to Boehringer Ingelheim do Brasil Quim. e Farm. Ltda. is bioequivalent to the Advil (R) and Alivium (R) formulations with regard to both the rate and the extent of absorption.

Drug Excipient Interaction Study with Polymorphic Forms of Tibolone

Powder mixtures (1:1) of tibolone polymorphic forms I (monoclinic) and II (triclinic) and excipients have been prepared and compacted. The samples were stored at 50 degrees C and 90% RH for one month and subsequently were evaluated using differential scanning calorimetry (DSC) and high-performance liquid chromatography (HPLC). The results indicate that during the compaction, the applied pressure reduced the chemical stability of tibolone in both polymorph forms. The triclinic form was more chemically unstable, both pure and in contact with excipients, than the monoclinic form. Lactose monohydrate was shown to reduce chemical degradation for both forms. Ascorbyl palmitate was shown to affect the tibolone stability differently depending on the polymorphic form used.

Cassava wastewater as a substrate for the simultaneous production of rhamnolipids and polyhydroxyalkanoates by Pseudomonas aeruginosa

Glycerol, cassava wastewater (CW), waste cooking oil and CW with waste frying oils were evaluated as alternative low-cost carbon substrates for the production of rhamnolipids and polyhydroxyalkanoates (PHAs) by various Pseudomonas aeruginosa strains. The polymers and surfactants produced were characterized by gas chromatography-mass spectrophotometry (MS) and by high-performance liquid chromatography-MS, and their composition was found to vary with the carbon source and the strain used in the fermentation. The best overall production of rhamnolipids and PHAs was obtained with CW with frying oil as the carbon source, with PHA production corresponding to 39% of the cell dry weight and rhamnolipid production being 660 mg l(-1). Under these conditions, the surface tension of the culture decreased to 30 mN m(-1), and the critical micelle concentration was 26.5 mg l(-1). It would appear that CW with frying oil has the highest potential as an alternative substrate, and its use may contribute to a reduction in the overall environmental impact generated by discarding such residues.

Caracterização química da erva mate (Ilex paraguariensis) : aplicação de diferentes processos de extração e influência das condições de plantio sobre a composição química

O objetivo deste trabalho foi estudar a composição química das folhas de erva-mate, sob diferentes condições agronômicas e técnicas de extração. Os métodos de extração usados foram maceração, ultra-som, extração com líquido pressurizado e extração com fluído supercrítico. Foram investigadas as variáveis que podem influenciar no processo de extração, tais como temperatura, pressão, polaridade do solvente, tempo de extração, massa de amostra, entre outras. A identificação dos compostos foi realizada por cromatografia gasosa acoplada à espectrometria de massas. Todos os métodos de extração utilizados mostraram-se eficientes para a obtenção dos extratos, com as diferenças sendo mais quantitativas do que qualitativas . Entre os métodos de extração que utilizam solventes orgânicos, a extração com líquido pressurizado mostrou-se mais eficiente, produzindo maior rendimento em massa de extrato e maior concentração de alguns dos compostos de interesse, com as vantagens de redução de solvente e tempo de extração. A composição química da erva-mate é influenciada pelas condições agronômicas de plantio, bem como pelas condições de extração de suas folhas. A melhor condição agronômica avaliada, ou seja, aquela que produziu maior quantidade de extrato, foi o cultivo das plantas a pleno sol, adubadas com nitrogênio e com idade de poda de 18 meses. A variável mais importante das técnicas de extração utilizadas foi a polaridade do solvente. Solventes de maior polaridade produziram maior rendimento em extrato. A análise cromatográfica dos extratos obtidos permitiu identificar cerca de 50 compostos qualitativamente e 6 quantitativamente, destacando-se a cafeína, fitol, ácido palmítico, ácido esteárico, esqualeno e vitamina E.

Non-invasive strategy in assessing asthma through biofluids metabolomics exploration: exhaled breath and urine potentialities

Asthma is a significant health issue in the pediatric population with a noteworthy growth over the years. The proposed challenge for this PhD thesis was the development of advanced methodologies to establish metabolomic patterns in urine and exhaled breath associated with asthma whose applicability was subsequently exploited to evaluate the disease state, the therapy adhesion and effect and for diagnostic purposes. The volatile composition of exhaled breath was studied combining headspace solid phase microextraction (HS-SPME) with gas chromatography coupled to mass spectrometry or with comprehensive two-dimensional gas chromatography coupled to mass spectrometry with a high resolution time of flight analyzer (GC×GC–ToFMS). These methodologies allowed the identification of several hundred compounds from different chemical families. Multivariate analysis (MVA) led to the conclusion that the metabolomic profile of asthma individuals is characterized by higher levels of compounds associated with lipid peroxidation, possibly linked to oxidative stress and inflammation (alkanes and aldehydes) known to play an important role in asthma. For future applications in clinical settings a set of nine compounds was defined and the clinical applicability was proven in monitoring the disease status and in the evaluation of the effect and / or adherence to therapy. The global volatile metabolome of urine was also explored using an HSSPME/GC×GC–ToFMS method and c.a. 200 compounds were identified. A targeted analysis was performed, with 78 compounds related with lipid peroxidation and consequently to oxidative stress levels and inflammation. The urinary non-volatile metabolomic pattern of asthma was established using proton nuclear magnetic resonance (1H NMR). This analysis allowed identifying central metabolic pathways such as oxidative stress, amino acid and lipid metabolism, gut microflora alterations, alterations in the tricarboxylic acid (TCA) cycle, histidine metabolism, lactic acidosis, and modification of free tyrosine residues after eosinophil stimulation. The obtained results allowed exploring and demonstrating the potential of analyzing the metabolomic profile of exhaled air and urine in asthma. Besides the successful development of analysis methodologies, it was possible to explore through exhaled air and urine biochemical pathways affected by asthma, observing complementarity between matrices, as well as, verify the clinical applicability.