Repositório Institucional - Unifesp :: Navegando por Palavras-chave "mass spectrometry"

Navegando por Palavras-chave "mass spectrometry"

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Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatus
(Amer Soc Biochemistry Molecular Biology Inc, 2007-08-01) Toledo, Marcos S. [UNIFESP]; Levery, Steven B.; Bennion, Beau; Guimaraes, Luciana L. [UNIFESP]; Castle, Sherry A.; Lindsey, Rebecca; Momany, Michelle; Park, Chaeho; Straus, Anita Hilda [UNIFESP]; Takahashi, Helio Kiyoshi [UNIFESP]; Univ New Hampshire; Universidade Federal de São Paulo (UNIFESP); Univ Georgia
Acidic glycosphingolipid components were extracted from the opportunistic mycopathogen Aspergillus fumigatus and identified as inositol phosphorylceramide and glycosylinositol phosphorylceramides (GIPCs). Using nuclear magnetic resonance sppectroscopy, mass spectrometry, and other techniques, the structures of six major components were elucidated as Ins-P-Cer (Af-0), Manp (alpha 1 -> 3) Manp(alpha 1 -> 2) Ins-P-Cer (Af-2), Manp(a1 -> 2) Manp (alpha 1 -> 3) Manp(alpha 1 -> 2) Ins-P-Cer (Af-3a), Manp(a1 -> 3) [Galf(beta 1 -> 6)] Manp(alpha 1 -> 2)-Ins-P-Cer (Af-3b), Manp (alpha 1 -> 2)-Manp(alpha 1 -> 3)[ Galf(beta 1 -> 6)] Manp(beta 1 -> 2) Ins-P-Cer (Af-4), and Manp(alpha 1 -> 3) Manp(alpha 1 -> 6) GlcpN(alpha 1 -> 2) Ins-P-Cer (Af-3c) ( where Ins 5 myo-inositol and P = phosphodiester). A minor A. fumigatus GIPC was also identified as the N-acetylated version of Af-3c (Af-3c*), which suggests that formation of the GlcN alpha 1 -> 2Ins linkage may proceed by a two-step process, similar to the GlcN alpha 1 -> 2Ins linkage in glycosylphosphatidylinositol (GPI) anchors (transfer of GlcNAc, followed by enzymatic de-N-acetylation). the glycosylinositol of Af-3b, which bears a distinctive branching Galf (b1Y6) residue, is identical to that of a GIPC isolated previously from the dimorphic mycopathogen Paracoccidioides brasiliensis (designated Pb-3), but components Af-3a and Af-4 have novel structures. Overlay immunostaining of A. fumigatus GIPCs separated on thinlayer chromatograms was used to assess their reactivity against sera from a patient with aspergillosis and against a murine monoclonal antibody (MEST-1) shown previously to react with the Galf (beta 1 -> 6) residue in Pb-3. These results are discussed in relation to pathogenicity and potential approaches to the immunodiagnosis of A. fumigatus.
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Caracterização proteômica, peptidômica e transcriptômica dos venenos de aranhas do gênero acanthoscurria
(Universidade Federal de São Paulo (UNIFESP), 2015-04-09) Abreu, Thiago Ferreira de [UNIFESP]; Tashima, Alexandre Keiji Tashima [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Spiders are one of the most successful animals on the planet, currently recording 45,330 species divided into 3957 genus and separated in two suborders: Mesothelae and Opistothelae. The most studied spiders belong to the second suborder, which are organized in two groups based on chelicerae position (Araneomorph and Mygalomorphae) and are studied, among other reasons, because of their medical and biological relevance. The chelicerae inoculates a toxic secretion produced by the venom gland for subduying preys and protection against predators. The venom presents a range of components with specific functions and synergical effects. Recent studies of spider venoms demonstrated the presence of proteins and peptides with antimicrobial activity against fungi, bacteria and protozoa, ion channel modulation and selective activity against vertebrate and invertebrate receptors, indicating the biotechnological potential for these molecules. The study of the spider venom molecular arsenal is also relevant to the understanding of phylogeny and evolutionary success and to determine variations in composition, which may depend on individual variability, sexual dimorfism and diet. Nonetheless, there are few studies about these animals. In this study, considering the biotechnological potential and the biological relevance of the toxins in spider venoms, a quantitative proteomic study of venoms from male and female spiders belonging to Acanthoscurria gomesiana and Acanthoscurria juruenicola species was developed, besides transcriptomics of A. juruenicola venom glands, and antimicrobial assays of purified peptides. Transcriptomic analysis of female A. juruenicola venom glands resulted in 88,335 protein sequences obtained by translation of its respective cDNAs. Digestion of venoms with trypsin and thermolysin followed by mass spectrometry analysis by data-dependent acquisition (DDA) and data-independent acquisition (DIA) approaches resulted in the identification of 353 proteins in A. juruenicola venoms and 188 proteins in A. gomesiana venoms by automated de novo sequencing and database searches. Absolute quantification of proteins present in A. juruenicola spider venoms by DIA showed a prevalence of toxins, such as cysteine-rich venom protease (CRISPs), theraphotoxins, metalloendopeptidases, lipases, hyaluronidases, tyrosine-phosphatase and metallocarboxypeptidases. One of A. juruenicola theraphotoxins showed complete homology with the peptide already described from Acanthoscurria paulensis venom (U1-theraphotoxin-Ap1a) and a new theraphotoxin from A. gomesiana, Ag1a, was fully characterized. Crude venom fractionation by gel filtration chromatography in order to purify peptides (1-7 kDa) was accomplished, enabling the isolation and characterization of A. gomesiana and A. juruenicola theraphotoxins. Antimicrobial assays against a yeast (Candida albicans) and three bacteria, two Gram negatives (Pseudomonas aeruginosa and Escherichia coli) and one Gram positive (Micrococcus luteus) was accomplished with these peptides and the minimum inhibitory concentrations (MIC) were determined.
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Caracterização proteômica, peptidômica e transcriptômica dos venenos de aranhas do gênero acanthoscurria
(Universidade Federal de São Paulo (UNIFESP), 2015-04-09) Abreu, Thiago Ferreira de [UNIFESP]; Tashima, Alexandre Keiji Tashima [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Spiders are one of the most successful animals on the planet, currently recording 45,330 species divided into 3957 genus and separated in two suborders: Mesothelae and Opistothelae. The most studied spiders belong to the second suborder, which are organiz
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Characterization of Novel O-Glycans Isolated from Tear and Saliva of Ocular Rosacea Patients
(Amer Chemical Soc, 2013-03-01) Ozcan, Sureyya; An, Hyun Joo; Vieira, Ana C. [UNIFESP]; Park, Gun Wook; Kim, Jae Han; Mannis, Mark J.; Lebrilla, Carlito B.; Univ Calif Davis; Chungnam Natl Univ; Univ Calif; Universidade Federal de São Paulo (UNIFESP)
O-Glycans in saliva and tear isolated from patients suffering from ocular rosacea, a form of inflammatory ocular surface disease, were profiled, and their structures were elucidated using high resolution mass spectrometry. We have previously shown that certain structures, particularly sulfated oligosaccharides, increased in the tear and saliva of rosacea patients in this study, the structures of these glycans were elucidated using primarily tandem mass spectrometry. There were important similarities in the glycan profiles of tears and saliva with the majority of the structures in common. the structures of the most abundant species common to both tear and saliva, which were also the most abundant species in both, were elucidated. for sulfated species, the positions of the sulfate groups were localized. the majority of the structures were new, with the sulfated glycans comprising mucin core 1- and core 2-type structures. As both saliva and tear are rich in mucins, it is suggested that the O-glycans are mainly components of mucins. the study further illustrates the strong correspondence between the glycans in the tear and saliva of ocular rosacea patients.
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Comparative Proteomic Analysis of Lysine Acetylation in Trypanosomes
(Amer Chemical Soc, 2018) Moretti, Nilmar Silvio [UNIFESP]; Cestari, Igor; Anupama, Atashi; Stuart, Ken; Schenkman, Sergio [UNIFESP]
Protein acetylation is a post-translational modification regulating diverse cellular processes. By using proteomic approaches, we identified N-terminal and epsilon-lysine acetylated proteins in Trypanosoma cruzi and Trypanosoma brucei, which are protozoan parasites that cause significant human and animal diseases. We detected 288 lysine acetylation sites in 210 proteins of procyclic form, an insect stage of T. brucei, and 380 acetylation sites in 285 proteins in the form of the parasite that replicates in mammalian bloodstream. In T. cruzi insect proliferative form we found 389 epsilon-lysine-acetylated sites in 235 proteins. Notably, we found distinct acetylation profiles according to the developmental stage and species, with only 44 common proteins between T. brucei stages and 18 in common between the two species. While K-ac proteins from T. cruzi are enriched in enzymes involved in oxidation/reduction balance, required for the parasite survival in the host, in T. brucei, most K-ac proteins are enriched in metabolic processes, essential for its adaptation in its hosts. We also identified in both parasites a quite variable N-terminal acetylation sites. Our results suggest that protein acetylation is involved in differential regulation of multiple cellular processes in Trypanosomes, contributing to our understanding of the essential mechanisms for parasite infection and survival.
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Determinação do perfil lipídico metabolômico plasmático, por espectrometria de massas, de pacientes com neoplasias mieloproliferativas crônicas, síndromes mielodisplásicas e leucemia mieloide aguda
(Universidade Federal de São Paulo (UNIFESP), 2015-03-25) Oliveira, Adriana Ramos de [UNIFESP]; Chauffaille, Maria de Lourdes Lopes Ferrari Chauffaille [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Introduction: There are thousands of individual lipid species in the cells interacting in different compartments in the cell membrane. The functional consequences of this diversity are not yet fully understood and new technological tools are being developed with the aim of a more comprehensive investigation. Over the past two decades, mass spectrometry (MS) has emerged as the main method used in lipidomics analysis, which allows the structural characterization and quantification of complex lipids and their metabolites. Due to the importance of this field we have considered the use of the lipidomic innovative platform to identify differences in the plasma lipid metabolomic profile of hematological patients with Myeloid Neoplasms. Purpose: Determine the lipid metabolomic profile comparative of blood plasma samples from healthy individuals and patients with Myeloproliferative Neoplasms, Myelodysplastic Syndromes and Acute Myeloid Leukemia and evaluate the existence of possible biomarkers. Methods: Untargeted Shotgun MS/MS Analysis was performed from plasma samples from 153 participants were analyzed being, 90 of the Control Group, 43 Myeloproliferative Neoplasms, 11 Myelodysplastic Syndromes and 9 Acute Myeloid Leukemias. Data were acquired using the AB-Sciex Analyst TF, processed using the AB-Sciex LipidView? and the web-based analytical pipeline MetaboAnalyst 2.0. Results: Untargeted analysis identified in negative and positive-modes a total of 658 features at 2 ppm resolution. PCA and PLS-DA analysis revealed clear discrimination among groups, in particular for AML patients. Main lipid groups differentially expressed were: Monoacylglycerols (MAG), Glucosylceramide E (GlcdE), Ethyl Esters (EE), Lysophosphatidic acid (LPA), Sulfoquinovosil diacylglycerols (SQDG), Monoglycerols (MG), Methyl Ethanolamines (ME), Lysophosphatidylcholines (LPC), Dimethyl Phosfatidyletanilamines (DMPE), Monometylphosphatidiletanolamines (MMPE), Ceramide-1-phosphate (CerP), Glicerophosphoglycerols (GP), Lysomonomethyl-Phosphatidyl ethanolamines (LMMPE), Phosphatidic Acids (PA), Ergosterols (ERG), Glycerophosphoserine (PS), Diacylglycerols (DAG), Hexocylceramides (HexCer) and Lanosterol (Lan). ROC Curve Analysis revealed Total LMMPE as the strongest discriminating marker between Controls from Patients with MDS or AML (Sensitivity= 0.95 (0.824-1); Specificity= 0.8941 (0.847-0953); Positive Likelihood Ratio= 8.972 and Negative Likelihood Ratio =0.05592 and T Test= 7.576E-12). In addition these lipids were also able to differentiate MDS and AML from NMP (Sensitivity= 0.9118 (0.824-1), Specificity= 0.95 (0.85-1); Positive Likelihood Ratio= 18.2 and Negative Likelihood Ratio= 0.05592). Conclusions: The Myeloproliferative Neoplasms from the point of view of global plasma lipidomics are accompanied by several modifications. In particular the Lysomonomethyl-Phosphatidyl ethanolamines seems to play important differentiating roles among them.
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Development and validation of a rapid and sensitive LC-ESI-MS/MS method for ondansetron quantification in human plasma and its application in comparative bioavailability study
(Wiley-Blackwell, 2010-11-01) Moreira, Roberto F.; Salvadori, Myriam C.; Azevedo, Cristina P.; Oliveira-Silva, Diogo [UNIFESP]; Borges, Diego C.; Moreno, Ronlison A.; Sverdloff, Carlos E.; Borges, Ney C.; Universidade Estadual de Campinas (UNICAMP); ChromAnal MCM Anal Labs; Universidade Federal de São Paulo (UNIFESP); Fac Med; Synchrophar Assessora & Desenvolvimento Projetos
The validation of a high throughput and specific method using a high-performance liquid chromatography coupled to electrospray (ES+) ionization tandem triple quadrupole mass spectrometric (LC-ESI-MS/MS) method for ondansetron quantification in human plasma is described Human plasma samples were extracted by liquid-liquid extraction (LLE) using methyl tert-butyl ether and analyzed by LC-ESI-MS/MS the limit of quantification was 0 2 ng/mL and the method was linear in the range 0 2-60 ng/mL the intra-assay precisions ranged from 1 6 to 7 7%, while inter-assay precisions ranged from 2 1 to 5 1% the intra-assay accuracies ranged from 97 5 to 108 2%, and the inter-assay accuracies ranged from 97 3 to 107 0% the analytical method was applied to evaluate the relative bioavailability of two pharmaceutical formulations containing 8 mg of ondansetron each in 25 healthy volunteers using a randomized, two-period crossover design the geometric mean and respective 90% confidence interval (Cl) of ondansetron test/reference percent ratios were 90 15% (81 74-99 44%) for C(max) and 93 11% (83 01-104 43%) for AUC(o-t) Based on the 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) and AUC(o-inf), , it was concluded that the test formulation is bioequivalent to the reference one with respect to the rate and extent of absorption of ondansetron Copyright (C) 2010 John Wiley & Sons, Ltd
Acesso aberto (Open Access)
Development of nanoinjector devices for electrospray ionization - tandem mass spectrometry (ESI-MSn)
(Sociedade Brasileira de Química, 2012-09-01) Assunção, Nilson Antonio [UNIFESP]; Nakayasu, Ernesto Satoshi; Daffre, Sirlei; Carrilho, Emanuel; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
In mass spectrometric (MS) systems with electrospray ionization (ESI), the sample can be analyzed coupled to separation systems (such as liquid chromatography or capillary electrophoresis) or simply by direct infusion. The greatest benefit of the type of injection is the possibility of continuous use of small amounts of samples over a long period of time. This extended analysis time allows a complete study of fragmentation by mass spectrometry, which is critical for structure elucidation of new compounds, or when using an ion trap mass analyzer. The injector filled with the sample is placed at the ESI source inlet creating an electric field suitable for the continuous formation of a spray (solvent and sample) and consequently, the gradual and even release of the sample. For the formation of the spray, is necessary that the injector end is metalized. The formation of a bilayer of titanium and gold provided an excellent attachment of the film, resulting in a nanoinjector for ionization/spray formation in the system for MS. The nanoinjectors showed high repeatability and stability over 100 min by continuous sampling with 10 µL of sample.
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Dimorphic expression of cerebrosides in the mycopathogen Sporothrix schenckii
(Lipid Research Inc, 2000-05-01) Toledo, Marcos S. [UNIFESP]; Levery, Steven B.; Straus, Anita H. [UNIFESP]; Takahashi, Helio K. [UNIFESP]; Univ Georgia; Universidade Federal de São Paulo (UNIFESP)
Major neutral glycosphingolipid components were extracted from Sporothrix schenckii, a dimorphic fungus exhibiting a hyphal saprophytic phase and a yeast parasitic phase responsible for chronic mycotic infections in mammalian hosts. These components, one from the mycelial form and two from the yeast form, were purified and their structures were elucidated by H-1 nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and tandem ESI-MS/MS, All three were characterized as cerebrosides (monohexosylceramides) containing (4E, 8E)-9-methyl-4,8-sphingadienine as the long-chain base attached to N-2'-hydroxyoctadecanoate and N2'-hydroxy-(E)-Delta(3)-octadecenoate as the fatty acyl components. However, while the mycelial form expressed only beta-glucopyranosylceramide, the yeast form expressed both beta-gluco- and beta-galactopyranosylceramides in approximately equal amounts. In addition, while the glucosylceramides of both mycelial and yeast forms had similar proportions of saturated and (E)-Delta(3) unsaturated 2-hydroxy fatty acid, the galactocerebroside of the yeast form had significantly higher levels of (E)Delta(3) unsaturation. The differences in cerebroside hexose structure represent a novel type of glycosphingolipid dimorphism not previously reported in fungi, Possible implications of these findings with respect to regulation of morphological transitions in S, schenckii and other dimorphic fungi are discussed. Dimorphic expression of cerebrosides in the mycopathogen Sporothrix schenckii.
Acesso aberto (Open Access)
Electrospray ionization mass spectrometry applied to study the radical acetylation of amino acids, peptides and proteins
(Sociedade Brasileira de Química, 2013-12-01) Alves, Atecla Nunciata Lopes [UNIFESP]; Jedlicka, Leticia Dias Lima [UNIFESP]; Massari, Júlio; Juliano, Maria Aparecida [UNIFESP]; Bechara, Etelvino José Henriques [UNIFESP]; Assunção, Nilson Antonio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
Recently, our group proposed a process that generated acetyl radicals in a reaction medium buffered with a diacetyl/peroxynitrite system. Diacetyl is a flavoring agent in food, cigarettes and drinks. Peroxynitrite is found in mitochondria, and in certain conditions, such as an infection in humans, its concentration is augmented significantly. In biological systems, radical compounds can easily modify the structure and activity of nucleic acids, proteins and other biomolecules, causing significant oxidative stress. Based on paramagnetic resonance and mass spectrometry data, this work discusses products that prove acetyl radicals are produced and are able to form stable covalent bonds with amino acid (acetylated products), peptide and protein adducts. These materials were separated and detected by capillary electrophoresis coupled with tandem mass spectrometry or offline mass spectrometry. The reaction medium contained a 1:2 mixture of diacetyl and peroxynitrite dissolved in 200 mmol L-1 of pH 7.2 sodium phosphate buffer. These experiments also reveal the double acetylation of lysine, demonstrating the high reactivity of these compounds when in contact with nitrogen-containing biomolecules readily found in biological systems. These structural changes might be an epigenetic source of post-translational protein modification.
Acesso aberto (Open Access)
Evaluation of MALDI-TOF MS in the microbiology laboratory
(Sociedade Brasileira de Patologia ClínicaSociedade Brasileira de PatologiaSociedade Brasileira de Citopatologia, 2013-06-01) Santos, Anderson Fernandes; Cayô, Rodrigo [UNIFESP]; Schandert, Lygia [UNIFESP]; Gales, Ana Cristina [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); National Council for Scientific and Technological Development; Brazilian Society of Infectious Diseases Bacteriology Committee
Rapid identification of microorganisms by the clinical microbiology laboratory is of crucial importance for optimal patients’ management and treatment. In general, bacterial identification by conventional methods requires 18-24 hours for colony isolation and at least 24 additional hours for species identification. New technologies in microbiology have focused on the rapid diagnosis of bloodstream infections, since they are associated with high morbidity and mortality rates.
Acesso aberto (Open Access)
Homemade Capillary Electrophoresis Coupled to a Mass Spectrometer
(Taylor & Francis Inc, 2015-01-02) Moraes, Janderson Aparecido de [UNIFESP]; Carrilho, Emanuel; Assunção, Nilson Antonio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
A system was developed in our laboratory to couple capillary electrophoresis with mass spectrometry. the capillary electrophoresis system was equipped with a high voltage supply, and a microcontroller with assembly language programming was developed for the computational control of the system. the MS system was a commercial Thermo Finningan LCQ ion trap mass spectrometer. the robustness of the coupled system was evaluated using standard protein samples (lysozyme, aprotinin, and bovine albumin) and tryptic digests of lysozyme. the system showed positive results in terms of robustness, allowing for the separation of digested proteins and the identification of 33% of the total amino acids in a protein (6 of the 18 expected peptides). the limit of detection was in the order of 1 picomole (signal-to-noise ratio), which was considered satisfactory for this system. the system shows high versatility in tandem coupling and combinations with other analytical procedures.
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A influência do índice de massa corporal na fisiologia do folículo ovariano em ciclos de reprodução assistida
(Universidade Federal de São Paulo (UNIFESP), 2013-04-03) Porciuncula, Patricia Marafon [UNIFESP]; Bertolla, Ricardo Pimenta Bertolla [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Objective: To observe the influence of Body Mass Index (BMI) on the follicular metabolism of women submitted to controlled ovarian hyperstimulation. Methods: A prospective study was carried out including 129 follicular fluid samples from women submitted to controlled ovarian hyperstimulation and classified, according to BMI, in three groups according to the World Health Organization: eutrophic (20 to <25kg/m2), overweight (25 to <30kg/m2), and obese (>30kg/m2). Follicular fluid samples were obtained by ultrasound-guided transvaginal ovarian aspiration following controlled ovarian hyperstimulation. Immediately after collection, the follicular fluid samples were centrifuged to remove cellular debris and frozen until analysis. For quantification of the steroid hormones estradiol, progesterone, testosterone, androstenedione, and cortisol, tandem mass spectrometry was utilized (ID-LC-MS/MS). For proteomic analysis, three pools were formed (one for each group, normalized to total protein in each sample), and proteins were quantified and identified utilizing the shotgun proteomics approach nanoUPLC-nanoESI-MSE, resulting in ?Identified? and ?Quantified and Identified? proteins. Proteins observed in only one replicate were excluded from the study. For steroid profiling, groups were compared using one-way ANOVA followed by a Least Significant Differences posthoc test, or using a Kruskal-Wallis followed by a Mann-Whitney test. For the proteomics experiment a Kruskal-Wallis test was used, followed by a Mann-Whitney test. An alpha of 5% was adopted in the study. Protein lists were utilized for functional enrichment analysis. Results: Regarding the steroid profile, differences were observed between the BMI groups. The obesity group presented lower progesterone levels and higher androstenedione levels when compared to the other two groups. Also, estradiol, testosterone, and cortisol were higher in both overweight and obese groups when compared to eutrophic controls. Regarding proteomics, 284 proteins were identified, of which 152 were quantified. Major enriched functions were related to immune response, inflammation and coagulation. Moreover, seven proteins were hyperexpressed only in the obese patients. Finally, 20 proteins were exclusively expressed in the obesity group, 10 in the overweight group, and 19 in the eutrophic group. Conclusion: Follicular fluid metabolism is altered due to obesity in patients submitted to controlled ovarian hyperstimulation. The follicular fluid of overweight and obese patients presents altered steroid profiles, with increased estradiol, testosterone, and cortisol, and obese patients present increased androstenedione and decreased progesterone. Also, overweightness and obesity alter the follicular fluid protein profile of patients submitted to controlled ovarian hyperstimulation, with enriched immune and inflammatory response functions.
Acesso aberto (Open Access)
Leishmania (Viannia) braziliensis Inositol Phosphorylceramide: Distinctive Sphingoid Base Composition
(Frontiers Media Sa, 2017) De Castro Levatti, Erica V. [UNIFESP]; Toledo, Marcos S. [UNIFESP]; Costa, Renata Watanabe [UNIFESP]; Bahia, Diana [UNIFESP]; Mortara, Renato A. [UNIFESP]; Takahashi, Hello K. [UNIFESP]; Straus, Anita H. [UNIFESP]
Inositol phosphorylceramide (IPC), the major sphingolipid in the genus Leishmania but not found in mammals, is considered a potentially useful target for chemotherapy against leishmaniasis. Leishmania (Viannia) braziliensis is endemic in Latin America and causes American tegumentary leishmaniasis. We demonstrated that IPCs are localized internally in parasites, using a specific monoclonal antibody. Treatment with 5 mu M myriocin (a serine palmitoyltransferase inhibitor) rendered promastigotes 8-fold less infective than controls in experimental hamster infection, as determined by number of parasites per inguinal lymph node after 8 weeks infection, suggesting the importance of parasite IPC or sphingolipid derivatives in parasite infectivity or survival in the host. IPC was isolated from promastigotes of three L. (V.) braziliensis strains and analyzed by positive- and negative-ion ESI-MS. The major IPC ions were characterized as eicosasphinganine and eicosasphingosine. Negative-ion ESI-MS revealed IPC ion species at m/z 778.6 (d20:1/14:0), 780.6 (d20:0/14:0), 796.6 (t20:0/14:0), 806.6 (d20:1/16:0), and 808.6 (d20:0/16:0). IPCs isolated from L. (V) braziliensis and L. (L.) major showed significant differences in IPC ceramide composition. The major IPC ion from L. (L.) major detected in negative-ion ESI-MS at m/z 780.6, was composed of ceramide d16:1/18:0. Our results suggest that sphingosine synthase (also known as serine palmitoyltransferase
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Low molecular weight squash trypsin inhibitors from Sechium edule seeds
(Elsevier B.V., 2006-02-01) Laure, H. J.; Faca, V. M.; Izumi, C.; Padovan, J. C.; Greene, L. J.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP); Rockefeller Univ
Nine chromatographic components containing trypsin inhibitor activity were isolated from Sechium edule seeds by acetone fractionation, gel filtration, affinity chromatography and RP-HPLC in an overall yield of 46% of activity and 0.05% of protein. the components obtained with highest yield of total activity and highest specific activity were sequenced by Edman degradation and their molecular masses determined by mass spectrometry. the inhibitors contained 31, 32 and 27 residues per molecule and their sequences were: SETI-IIa, EDRKCPKILMRCKRDSDCLAKCTCQESGYCG; SETI-IIb, EEDRKCPKILMRCKRDSDCLAKCTCQESGYCG and SETI-V, CPRILMKCKLDTDCFPTCTCRPSGFCG. SETI-IIa and SETI-IIb, which differed by an amino-terminal E in the IIb form, were not separable under the conditions employed. the sequences are consistent with consensus sequences obtained from 37 other inhibitors: CPriIlmeCk_DSDCla_C_C_G_CG, where capital letters are invariant amino acid residues and lower case letters are the most preserved in this position. SETI-II and SETI-V form complexes with trypsin with a 1: 1 stoichiometry and have dissociation constants of 5.4 x 10(-11) M and 1.1 x 10(-9) M, respectively. (c) 2005 Elsevier B.V. All rights reserved.
Acesso aberto (Open Access)
Morpho/Proteomic Comparative between High Grade Pleomorphic Sarcoma and Metastasis Diagnosed in an Old Captive Common Hippo
(Soc Brasileira Quimica, 2018) Silva, Adriana R. [UNIFESP]; de Lacerda, Jose T. J. G. [UNIFESP]; Faria, Bernadete [UNIFESP]; da Cunha, Isabela W.; de Andrade, Vitor P.; Assuncao, Nilson A. [UNIFESP]
Old age is a risk factor for cancer development in humans and animals, and studies have shown that tumors in animals are acceptable models for studying human cancers, considering the similarities between their factors. This work was conducted in a 53-year-old captive female common hippo (Hippopotamus amphibious) with a left leg tumor and metastatic mass. Histopathological and immunohistochemical analyses were carried out with a final diagnosis of a high grade pleomorphic sarcoma. A proteomic study using mass spectrometry was added in order to identify further aspects of the primary tumor and metastasis which could improve our understanding, and each tissue showed a proteomic profile indicative of its pathologic state with significant differences between healthy tissue, primary and metastatic tumors. Low levels of beta-actin in primary tumors were identified, and this may be associated with a possible consequence of cytoskeleton dynamic modification. In metastatic tissue, these dynamics may be affected by the presence of HSP chaperone 60.
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Non-invasive prediction of blastocyst implantation, ongoing pregnancy and live birth, by mass spectrometry lipid fingerprinting
(Soc Brasileira Reproducao Assistida-Sbra, 2016) Borges, Edson, Jr.; Braga, Daniela Paes Almeida Ferreira [UNIFESP]; Setti, Amanda Souza; Montani, Daniela Antunes [UNIFESP]; Cabral, Elaine Cristina; Eberlin, Marcos N.; Lo Turco, Edson Guimaraes [UNIFESP]; Iaconelli, Assumpto, Jr.
Objective: To identify lipid markers of blastocyst implantation and ongoing pregnancy by day three culture medium mass spectrometry (MS) fingerprinting. Methods: For this study, 33 culture media samples were harvested on day three, from 22 patients undergoing day five embryo transfers. All embryos achieved the blastocyst stage and were split into groups based on their implantation (Negative Implantation, n= 14 and Positive Implantation, n= 19). The positive implantation cycles resulted in successful ongoing pregnancies. The lipid extraction was performed by the Bligh-Dyer protocol and mass spectra were obtained with a direct infusion into a Q-Tof mass spectrometer. The data obtained was analyzed by Principal Component Analysis (PCA) and Partial Least Square Discrimination Analysis (PLS-DA). The statistical analysis was performed using the Metabo-Analyst 2.0. Results: The variable importance in the projection (VIP) plot of the PLS-DA provided a list of four ions, in the positive mode, with an area under the curve (AUC) of 73.5%
Acesso aberto (Open Access)
Peptide Structure Modifications: Effect of Radical Species Generated by Controlled Gamma Ray Irradiation Approach
(Pharmaceutical Soc Japan, 2013-04-01) Vieira, Renata de Freitas Fischer [UNIFESP]; Nardi, Daniela Teves [UNIFESP]; Nascimento, Nanci; Rosa, Jose Cesar [UNIFESP]; Nakaie, Clovis Ryuichi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
The present work aimed at evaluating the radiolysis effect upon a set of peptides, most of them involved in physiological functions. To generate reactive radical species, a Co-60 source (up to 15 kGy) was used for controlled gamma irradiation of some peptide solutions including derivatives attaching the stable free radical Toac (2,2,6,6-tetramethypiperidine-1-oxyl-4-amino-4-carboxylic acid). Regardless of the peptide sequence, a nonlinear and progressive degradation of a total of nine peptides was detected. The results were interpreted in the light of the half-life dose (D-1/2) parameter which represents the dose necessary for 50% peptide structure degradation. The vasoactive angiotensin II (AngII)'s analogue Ang-(1-7) showed greater stability towards gamma ray radiation than bradykinin (BK), Toac(0)-BK, Pro(4)-BK (D-1/2 around 4 and 2 kGy, respectively) which decreased to about 0.5-1.0 kGy in the case of acetyl-alpha-melanocyte-stimulating hormone (Aca-MSH) and substance P (SP). In terms of peptide structural modifications, the data acquired from different analytical methods suggested a Phe to Tyr (or its ortho and/or meta isomers) transformation as a consequence of the hydroxyl moiety insertion. Noteworthy, this effect seemed to be position-dependent as only Phe located at or near the C-terminal portion seemed to display this transformation. In contrast, Met is comparatively more easily oxidized, thus allowing to conclude that gamma irradiation may induce a complex position and/or sequence-dependent effect on peptides. As previously applied for BK, some irradiated peptides were submitted to their by-products purification, indeed a complementary target of the present approach for development of uncommon analogues for further structure-function investigation.
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Post-translational modifications of Trypanosoma cruzi histone H4
(Elsevier B.V., 2006-12-01) Chagas da Cunha, Julia Pinheiro; Nakayasu, Ernesto Satoshi; Almeida, Igor Correia de; Schenkman, Sergio; Universidade Federal de São Paulo (UNIFESP); Univ Texas
Histone tails provide sites for a variety of post-translational modifications implicated in the control of gene expression and chromatin assembly. As both histones and control of gene expression in trypanosomes are highly divergent compared to most eukaryotes, post-translational modifications of Trypanosoma cruzi histones were investigated. After in vivo incubation of live parasites with radiolabeled precursors, histone H4 mainly incorporates [H-3]-acetyl, and to a lesser extent [H-3]-methyl residues. in contrast, histone H3 preferentially incorporates [H-3]-methyl residues. the modifications of histone H4 were further characterized by mass spectrometry. MALDI-TOF-TOF-MS analysis revealed that peptides from histone H4 amino-terminus, obtained by either endoproteinase Glu-C or endoproteinase Arg-C digestion, contain isoforms with 14 and 42 Da additions, suggesting the presence of simultaneous acetylations and/or methylations. Tandem mass spectrometry analysis demonstrated that the N-terminal alanine is methylated, and lysine residues at positions 4, 10, 14 and 57 are acetylated; lysine at position 18 is mono-methylated, while arginine at position 53 is dimethylated. Immunoblotting analyses using specific antibodies raised against synthetic and acetylated peptides of T cruzi histone H4 indicate that lysine 4 is acetylated in the majority of histone H4, while other acetylations at the N-terminus portion of histone H4 are less abundant. (c) 2006 Elsevier B.V. All rights reserved.
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Protein SUMOylation is Involved in Cell-cycle Progression and Cell Morphology in Giardia lamblia
(Wiley, 2017) Di Genova, Bruno M. [UNIFESP]; da Silva, Richard C. [UNIFESP]; da Cunha, Julia P. C.; Gargantini, Pablo R.; Mortara, Renato A. [UNIFESP]; Tonelli, Renata R. [UNIFESP]
The unicellular protozoa Giardia lamblia is a food- and waterborne parasite that causes giardiasis. This illness is manifested as acute and self-limited diarrhea and can evolve to long-term complications. Successful establishment of infection by Giardia trophozoites requires adhesion to host cells and colonization of the small intestine, where parasites multiply by mitotic division. The tight binding of trophozoites to host cells occurs by means of the ventral adhesive disc, a spiral array of microtubules and associated proteins such as giardins. In this work we show that knock down of the Small Ubiquitin-like MOdifier (SUMO) results in less adhesive trophzoites, decreased cell proliferation and deep morphological alterations, including at the ventral disc. Consistent with the reduced proliferation, SUMO knocked-down trophozoites were arrested in G1 and in S phases of the cell cycle. Mass spectrometry analysis of anti-SUMO immunoprecipitates was performed to identify SUMO substrates possibly involved in these events. Among the identified SUMOylation targets, -tubulin was further validated by Western blot and confirmed to be a SUMO target in Giardia trophozoites.

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