Solid phase microextraction, mass spectrometry and metabolomic approaches for detection of potential urinary cancer biomarkers: a powerful strategy for breast cancer diagnosis

A sensitive assay to identify volatile organic metabolites (VOMs) as biomarkers that can accurately diagnose the onset of breast cancer using non-invasively collected clinical specimens is ideal for early detection. Therefore the aim of this study was to establish the urinary metabolomic profile of breast cancer patients and healthy individuals (control group) and to explore the VOMs as potential biomarkers in breast cancer diagnosis at early stage. Solid-phase microextraction (SPME) using CAR/PDMS sorbent combined with gas chromatography–mass spectrometry was applied to obtain metabolomic information patterns of 26 breast cancer patients and 21 healthy individuals (controls). A total of seventy-nine VOMs, belonging to distinct chemical classes, were detected and identified in control and breast cancer groups. Ketones and sulfur compounds were the chemical classes with highest contribution for both groups. Results showed that excretion values of 6 VOMs among the total of 79 detected were found to be statistically different (p < 0.05). A significant increase in the peak area of (−)-4-carene, 3-heptanone, 1,2,4-trimethylbenzene, 2-methoxythiophene and phenol, in VOMs of cancer patients relatively to controls was observed. Statiscally significant lower abundances of dimethyl disulfide were found in cancer patients. Bioanalytical data were submitted to multivariate statistics [principal component analysis (PCA)], in order to visualize clusters of cases and to detect the VOMs that are able to differentiate cancer patients from healthy individuals. Very good discrimination within breast cancer and control groups was achieved. Nevertheless, a deep study using a larger number of patients must be carried out to confirm the results.

Avaliação da atividade antimicrobiana de sistemas emulsionados contendo óleos naturais para o tratamento de infecções cutâneas

Natural oils have shown a scientific importance due to its pharmacological activity and renewable character. The copaiba (Copaifera langsdorffii) and Bullfrog (Rana catesbeiana Shaw) oils are used in folk medicine particularly because the anti-inflammatory and antimicrobial activities. Emulsion could be eligible systems to improve the palatability and fragrance, enhance the pharmacological activities and reduce the toxicological effects of these oils. The aim of this work was to investigate the antimicrobial activity of emulsions based on copaiba (resin-oil and essential-oil) and bullfrog oils against fungi and bacteria which cause skin diseases. Firstly, the essential oil was extracted from copaiba oil-resin and the oils were characterized by gas chromatography coupled to a mass spectrometry (GC-MS). Secondly, emulsion systems were produced. A microbiological screening test with all products was performed followed (the minimum inhibitory concentration, the bioautography method and the antibiofilm determination). Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Candida albicans, C. parapsilosis, C. glabrata, C. krusei and C. tropicalis American Type Culture Collection (ATCC) and clinical samples were used. The emulsions based on copaiba oil-resin and essential oil improved the antimicrobial activity of the pure oils, especially against Staphylococcus e Candida resistant to azoles. The bullfrog oil emulsion and the pure bullfrog oil showed a lower effect on the microorganisms when compared to the copaiba samples. All the emulsions showed a significant antibiofilm activity by inhibiting the cell adhesion. Thus, it may be concluded that emulsions based on copaiba and bullfrog oils are promising candidates to treatment of fungal and bacterial skin infections

Chemical Composition of Lipids Present in Cat and Dog Oocyte by Matrix-Assisted Desorption Ionization Mass Spectrometry (MALDI- MS)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The shielding effect of glycerol against protein ionization in electrospray mass spectrometry

Most commercial recombinant proteins used as molecular biology tools, as well as many academically made preparations, are generally maintained in the presence of high glycerol concentrations after purification to maintain their biological activity. The present study shows that larger proteins containing high concentrations of glycerol are not amenable to analysis using conventional electrospray ionization mass spectrometry (ESI-MS) interfaces. In this investigation the presence of 25% (v/v) glycerol suppressed the signals of Taq DNA polymerase molecules, while 1% (v/v) glycerol suppressed the signal of horse heart myoglobin. The signal suppression was probably caused by the interaction of glycerol molecules with the proteins to create a shielding effect that prevents the ionization of the basic and/or acidic groups in the amino acid side chains. To overcome this difficulty the glycerol concentration was decreased to 5% (v/v) by dialyzing the Taq polymerase solution against water, and the cone voltage in the ESI triple-quadrupole mass spectrometer was set at 80-130 V. This permitted observation of a mass spectrum that contained ions corresponding to protonation of up to 50% of the ionizable basic groups. In the absence of glycerol up to 85% of the basic groups of Taq polymerase became ionized, as observed in the mass spectrum at relatively low cone voltages. An explanation of these and other observations is proposed, based on strong interactions between the protein molecules and glycerol. For purposes of comparison similar experiments were performed on myoglobin, a small protein with 21 basic groups, whose ionization was apparently suppressed in the presence of 1% (v/v) glycerol, since no mass spectrum could be obtained even at high cone voltages. Copyright (C) 2003 John Wiley Sons, Ltd.

Analyzing glycerol-mediated protein oligomerization by electrospray ionization mass spectrometry

Glycerol is widely used as protein stabilizer, in both local and commercial preparations, so it has become necessary to develop methods for mass spectrometric analysis of protein preparations in the presence of glycerol. However, this stabilizing agent may cause signal suppression when present in high concentrations, and is also known to induce protein supercharging even at low concentrations. This work reports the,use of electrospray ionization (ESI) mass spectrometry to characterize glycerol-mediated protein oligomerization. this phenomenon seems to involve the formation of strong non-covalent interactions between protein and glycerol involving close contact between the monomers, leading to formation of protein oligomers adducted with glycerol molecules under the characteristic analytical conditions of the ESI interface. At high orders of oligomerization a lower number of glycerol molecules is required to maintain the high oligomeric states than for the dimers and trimers, and it is possible that for the higher oligomers the monomers become so close to one another that non-covalent bonds between the side chains of the amino acid residues in the proteins may be established. Copyright (C) 2005 John Wiley & Sons, Ltd.

Advantages of using nested collision induced dissociation/post-source decay with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: sequencing of novel peptides from wasp venom

We have examined the applicability of the 'nested' collision induced dissociation/post-source decay (CID/PSD) method to the sequencing of novel peptides from solitary wasps which have neurotoxic venom for paralyzing other insects. The CID/PSD spectrum of a ladder peptide derived from an exopeptidase digest was compared with that of the intact peptide. The mass peaks observed only in the CID/PSD spectrum of a ladder peptide were extracted as C-terminal fragment ions. Assignment of C-terminal fragment ions enabled calculation of N-terminal fragment masses, leading to differentiation between N-terminal fragment ions and internal fragment ions. This methodology allowed rapid and sensitive identification by removing ambiguity in the assignment of the fragment ions, and proved useful for sequencing unknown peptides, in particular those available as natural products with a limited supply. Copyright (C) 2000 John Wiley & Sons, Ltd.

Dynamics of glyphosate-induced conformational changes of Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19) determined by hydrogen-deuterium exchange and electrospray mass spectrometry

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the reaction between shikimate 3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate 3-phosphate, an intermediate in the shikimate pathway, which leads to the biosynthesis of aromatic amino acids. EPSPS exists in an open conformation in the absence of substrates and/or inhibitors and in a closed conformation when bound to the substrate and/or inhibitor. In the present report, the H/D exchange properties of EPSPS from Mycobacterium tuberculosis (Mt) were investigated for both enzyme conformations using ESI mass spectrometry and circular dichroism (CD). When the conformational changes identified by H/D exchanges were mapped on the 3-D structure, it was observed that the apoenzyme underwent extensive conformational changes due to glyphosate complexation, characterized by an increase in the content of alpha-helices from 40% to 57%, while the beta-sheet content decreased from 30% to 23%. These results indicate that the enzyme underwent a series of rearrangements of its secondary structure that were accompanied by a large decrease in solvent access to many different regions of the protein. This was attributed to the compaction of 71% of alpha-helices and 57% of beta-sheets as a consequence of glyphosate binding to the enzyme. Apparently, MtEPSPS undergoes a series of inhibitor-induced conformational changes, which seem to have caused synergistic effects in preventing solvent access to the core of molecule, especially in the cleft region. This may be part of the mechanism of inhibition of the enzyme, which is required to prevent the hydration of the substrate binding site and also to induce the cleft closure to avoid entrance of the substrates.

Monitoring the positioning of short polycationic peptides in model lipid bilayers by combining hydrogen/deuterium exchange and electrospray ionization mass spectrometry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hydrogen/deuterium exchange mass spectrometry for characterizing phosphoenolpyruvate-induced structural transitions in Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.1)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Direct and Simultaneous Determination of Cd Cu, and Se in Blood Samples by Graphite Furnace Atomic Absorption Spectrometry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Feasibility of internal standardization in the direct and simultaneous determination of As, Cu and Pb in sugar-cane spirits by graphite furnace atomic absorption spectrometry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Re-examination of the anion derivatives of isoflavones by radical fragmentation in negative electrospray ionization tandem mass spectrometry: experimental and computational studies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Metabolite fingerprint of "capim dourado" (Syngonanthus nitens), a basis of Brazilian handcrafts

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

GC Fingerprints Coupled to Pattern-Recognition Multivariate SIMCA Chemometric Analysis for Brazilian Gasoline Quality Studies

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Metabolic fingerprinting using direct flow injection electrospray ionization tandem mass spectrometry for the characterization of proanthocyanidins from the barks of Hancornia speciosa

Direct flow injection electrospray ionization ion trap tandem mass spectrometry (ESI-IT-MS/MS) was used to investigate the polyphenolic compounds present in an infusion from the barks of Hancornia speciosa Gom. (Apocynaceae), a native Brazilian plant popularly known as 'mangabeira', used as a source of nutrition and against gastric disorders. After a simple sample filtration pretreatment the characteristic fingerprint of the infusion was performed in negative ion ESI mode in a few minutes. At low capillary-voltage activation, the deprotonated molecules ([M-H](-)) were observed and using collision-induced dissociation the product ion spectra showed the presence of a homologous series of B-type proanthocyanidins, as well as another series containing their respective C-glycosylated derivatives, with a degree of polymerization from 1 up to 6 units of interlinked catechins. Therefore, direct flow injection allowed us to identify the key compounds without preparative isolation of the components. Copyright (C) 2007 John Wiley & Sons, Ltd.