Laboratorio Of Espectrometria Of Massas

Campinas, Brazil

Laboratorio Of Espectrometria Of Massas

Campinas, Brazil
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Genaro-Mattos T.C.,University of Sao Paulo | Appolinario P.P.,University of Sao Paulo | Mugnol K.C.U.,Federal University of ABC | Bloch Jr. C.,Laboratorio Of Espectrometria Of Massas | And 3 more authors.
Chemical Research in Toxicology | Year: 2013

Mitochondrial cholesterol has been reported to be increased under specific pathological conditions associated with enhanced oxidative stress parameters. In this scenario, cholesterol oxidation would be increased, leading to the production of reactive aldehydes, including cholesterol carboxyaldehyde (ChAld). By using SDS micelles as a mitochondrial mimetic model, we have demonstrated that ChAld covalently modifies cytochrome c (cytc), a protein known to participate in electron transport and apoptosis signaling. This mimetic model induces changes in cytc structure in the same way as mitochondrial membranes do. Tryptic digestion of the cytc-ChAld adduct followed by MALDI-TOF/TOF analyses revealed that modifications occur at Lys residues (K22) localized at cytc site L, a site involved in protein-protein and protein-membrane interactions. Interestingly, ChAld ligation prevented cytc detachment from liposomes even under high ionic strength conditions. Overall, it can be concluded that ChAld ligation to Lys residues at site L creates a hydrophobic tail at cytc, which promotes cytc anchoring to the membrane. Although not investigated in detail in this study, cytc adduction to cholesterol derived aldehydes could have implications in cytc release from mitochondria under apoptotic stimuli. © 2013 American Chemical Society.


PubMed | Federal University of São Paulo, Paulista University, Federal University of São Carlos, Laboratorio Of Espectrometria Of Massas and University of Sao Paulo
Type: | Journal: Molecular plant pathology | Year: 2016

Citrus canker is a plant disease caused by gram-negative bacteria from the genus Xanthomonas, and the most virulent species is Xanthomonas citri subsp.citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of periplasm-enriched fraction was performed for XAC cells grown in pathogenicity-inducing (XAM-M) and pathogenicity non-inducing (NB) media, using two-dimensional electrophoresis combined with liquid chromatography-tandem mass spectrometry. Among the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional upregulated proteins related to cellular envelope metabolism included glucose-1-phosphate thymidylyltransferase, dTDP-4-dehydrorhamnose 3,5-epimerase, and peptidyl-prolyl cis-trans isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real-time PCR analyses for transglycosylase and superoxide dismutase confirmed that these proteins are upregulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60 kDa chaperonin and glyceraldehyde-3-phosphate dehydrogenase were identified, suggesting the presence of post-translational modifications. We propose that substantial alterations in the cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defense against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates of virulence-related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes like hrpD6, hrpG, hrpB7, hpa1, and hrpX. The results present new potential targets against XAC to be investigated in further functional studies. This article is protected by copyright. All rights reserved.


PubMed | Fundacao Pio Xii Hospital Of Cancer Of Barretos, Laboratorio Of Espectrometria Of Massas and University of Sao Paulo
Type: Journal Article | Journal: Genes & cancer | Year: 2016

Despite great advance in multiple myeloma (MM) treatment since 2000s, it is still an incurable disease and novel therapies are welcome. Therefore, the purpose of this study was to explore MM plasma cells (MM-PC) proteome, in comparison with their normal counterparts (derived from palatine tonsils of normal donors, ND-PC), in order to find potential therapeutic targets expressed on the surface of these cells. We also aimed to evaluate the proteome of MM cell lines with different genetic alterations, to confirm findings obtained with primary tumor cells. Bone marrow (BM) samples from eight new cases of MM and palatine tonsils from seven unmatched controls were submitted to PC separation and, in addition to two MM cell lines (U266, RPMI-8226), were submitted to protein extraction for mass spectrometry analyses. A total of 81 proteins were differentially expressed between MM-PC and ND-PC - 72 upregulated and nine downregulated; U266 vs. RPMI 8226 cell lines presented 61 differentially expressed proteins - 51 upregulated and 10 downregulated. On primary tumors, bioinformatics analyses highlighted upregulation of protein biosynthesis machinery, as well as downregulation of immune response components, such as MHC class I and II, and complement receptors. We also provided comprehensive information about U266 and RPMI-8226 cell lines proteome and could confirm some patients findings.


PubMed | Laboratorio Of Espectrometria Of Massas, CTBE - Brazilian Bioethanol Science and Technology Laboratory, University of Sao Paulo and University of Campinas
Type: | Journal: Scientific reports | Year: 2015

The development and progression of oral cavity squamous cell carcinoma (OSCC) involves complex cellular mechanisms that contribute to the low five-year survival rate of approximately 20% among diagnosed patients. However, the biological processes essential to tumor progression are not completely understood. Therefore, detecting alterations in the salivary proteome may assist in elucidating the cellular mechanisms modulated in OSCC and improve the clinical prognosis of the disease. The proteome of whole saliva and salivary extracellular vesicles (EVs) from patients with OSCC and healthy individuals were analyzed by LC-MS/MS and label-free protein quantification. Proteome data analysis was performed using statistical, machine learning and feature selection methods with additional functional annotation. Biological processes related to immune responses, peptidase inhibitor activity, iron coordination and protease binding were overrepresented in the group of differentially expressed proteins. Proteins related to the inflammatory system, transport of metals and cellular growth and proliferation were identified in the proteome of salivary EVs. The proteomics data were robust and could classify OSCC with 90% accuracy. The saliva proteome analysis revealed that immune processes are related to the presence of OSCC and indicate that proteomics data can contribute to determining OSCC prognosis.


PubMed | Laboratorio Of Espectrometria Of Massas, Instituto Do Cancer Do Estado Of Sao Paulo, University of Campinas, University of Sao Paulo and University of Virginia
Type: Journal Article | Journal: Oncotarget | Year: 2016

Targeted proteomics has flourished as the method of choice for prospecting for and validating potential candidate biomarkers in many diseases. However, challenges still remain due to the lack of standardized routines that can prioritize a limited number of proteins to be further validated in human samples. To help researchers identify candidate biomarkers that best characterize their samples under study, a well-designed integrative analysis pipeline, comprising MS-based discovery, feature selection methods, clustering techniques, bioinformatic analyses and targeted approaches was performed using discovery-based proteomic data from the secretomes of three classes of human cell lines (carcinoma, melanoma and non-cancerous). Three feature selection algorithms, namely, Beta-binomial, Nearest Shrunken Centroids (NSC), and Support Vector Machine-Recursive Features Elimination (SVM-RFE), indicated a panel of 137 candidate biomarkers for carcinoma and 271 for melanoma, which were differentially abundant between the tumor classes. We further tested the strength of the pipeline in selecting candidate biomarkers by immunoblotting, human tissue microarrays, label-free targeted MS and functional experiments. In conclusion, the proposed integrative analysis was able to pre-qualify and prioritize candidate biomarkers from discovery-based proteomics to targeted MS.


PubMed | Federal University of Fluminense, Laboratorio Of Espectrometria Of Massas, Instituto Nacional Of Cancer Inca, Oswaldo Cruz Institute and University of Campinas
Type: | Journal: Journal of proteomics | Year: 2016

Oligodendrocytes produce and maintain the myelin sheath of axons in the central nervous system. Because misassembled myelin sheaths have been associated with brain disorders such as multiple sclerosis and schizophrenia, recent advances have been made towards the description of the oligodendrocyte proteome. The identification of splice variants represented in the proteome is as important as determining the level of oligodendrocyte-associated proteins. Here, we used an oligodendrocyte proteome dataset deposited in ProteomeXchange to search against a customized protein sequence file containing computationally predicted splice variants. Our approach resulted in the identification of 39 splice variants, including one variant from the GTPase KRAS gene and another from the human glutaminase gene family. We also detected the mRNA expression of five selected splice variants and demonstrated that a fraction of these have their canonical proteins participating in direct protein-protein interactions. In conclusion, we believe our findings contribute to the molecular characterization of oligodendrocytes and may encourage other research groups working with central nervous system disorders to investigate the biological significance of these splice variants. The splice variants identified in this study may encode proteins that could be targeted in novel treatment strategies and diagnostic methods.Several disorders of the central nervous system (CNS) are associated with misassembled myelin sheaths, which are produced and maintained by oligodendrocytes (OL). Recently, the OL proteome has been explored to identify key proteins and molecular functions associated with CNS disorders. We developed an innovative approach to select, with a higher level of confidence, a relevant list of splice variants from a proteome dataset and detected the mRNA expression of five selected variants: EEF1D, KRAS, MFF, SDR39U1, and SUGT1. We also described splice variants extracted from OL proteome data. Among the splice variants identified, some are from genes previously linked to CNS and related disorders. Our findings may contribute to oligodendrocyte characterization and encourage other research groups to investigate the biological role of splice variants and to improve current treatments and diagnostic methods for CNS disorders.


PubMed | Laboratorio Of Espectrometria Of Massas, University of Washington and Instituto Do Cancer Do Estado Of Sao Paulo
Type: Journal Article | Journal: Proteomics | Year: 2016

Head and neck cancers, including oral squamous cell carcinoma (OSCC), are the sixth most common malignancy in the world and are characterized by poor prognosis and a low survival rate. Saliva is oral fluid with intimate contact with OSCC. Besides non-invasive, simple, and rapid to collect, saliva is a potential source of biomarkers. In this study, we build an SRM assay that targets fourteen OSCC candidate biomarker proteins, which were evaluated in a set of clinically-derived saliva samples. Using Skyline software package, we demonstrated a statistically significant higher abundance of the C1R, LCN2, SLPI, FAM49B, TAGLN2, CFB, C3, C4B, LRG1, SERPINA1 candidate biomarkers in the saliva of OSCC patients. Furthermore, our study also demonstrated that CFB, C3, C4B, SERPINA1 and LRG1 are associated with the risk of developing OSCC. Overall, this study successfully used targeted proteomics to measure in saliva a panel of biomarker candidates for OSCC.


Carnielli C.M.,Laboratorio Of Espectrometria Of Massas | Winck F.V.,Laboratorio Of Espectrometria Of Massas | Paes Leme A.F.,Laboratorio Of Espectrometria Of Massas
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2015

Proteomics experiments often generate a vast amount of data. However, the simple identification and quantification of proteins from a cell proteome or subproteome is not sufficient for the full understanding of complex mechanisms occurring in the biological systems. Therefore, the functional annotation analysis of protein datasets using bioinformatics tools is essential for interpreting the results of high-throughput proteomics. Although large-scale proteomics data have rapidly increased, the biological interpretation of these results remains as a challenging task. Here we reviewed basic concepts and different programs that are commonly used in proteomics data functional annotation, emphasizing the main strategies focused in the use of gene ontology annotations. Furthermore, we explored the characteristics of some tools developed for functional annotation analysis, concerning the ease of use and typical caveats on ontology annotations. The utility and variations between different tools were assessed through the comparison of the resulting outputs generated for an example of proteomics dataset. © 2014 Elsevier B.V. All rights reserved.


Tavares R.,Instituto Nacional Of Cancer Inca | de Miranda Scherer N.,Instituto Nacional Of Cancer Inca | Pauletti B.A.,Laboratorio Of Espectrometria Of Massas | Araujo E.,Federal University of Mato Grosso do Sul | And 6 more authors.
Proteomics | Year: 2014

The mechanism of alternative splicing in the transcriptome may increase the proteome diversity in eukaryotes. In proteomics, several studies aim to use protein sequence repositories to annotate MS experiments or to detect differentially expressed proteins. However, the available protein sequence repositories are not designed to fully detect protein isoforms derived from mRNA splice variants. To foster knowledge for the field, here we introduce SpliceProt, a new protein sequence repository of transcriptome experimental data used to investigate for putative splice variants in human proteomes. Current version of SpliceProt contains 159 719 non-redundant putative polypeptide sequences. The assessment of the potential of SpliceProt in detecting new protein isoforms resulting from alternative splicing was performed by using publicly available proteomics data. We detected 173 peptides hypothetically derived from splice variants, which 54 of them are not present in UniprotKB/TrEMBL sequence repository. In comparison to other protein sequence repositories, SpliceProt contains a greater number of unique peptides and is able to detect more splice variants. Therefore, SpliceProt provides a solution for the annotation of proteomics experiments regarding splice isofoms. The repository files containing the translated sequences of the predicted splice variants and a visualization tool are freely available at http://lbbc.inca.gov.br/spliceprot. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


PubMed | Laboratorio Of Espectrometria Of Massas and University of Campinas
Type: Journal Article | Journal: Clinical science (London, England : 1979) | Year: 2016

EEF1D (eukaryotic translation elongation factor 1) is a subunit of the elongation factor 1 complex of proteins that mediates the elongation process during protein synthesis via enzymatic delivery of aminoacyl-tRNAs to the ribosome. Although the functions of EEF1D in the translation process are recognized, EEF1D expression was found to be unbalanced in tumours. In the present study, we demonstrate the overexpression of EEF1D in OSCC (oral squamous cell carcinoma), and revealed that EEF1D and protein interaction partners promote the activation of cyclin D1 and vimentin proteins. EEF1D knockdown in OSCC reduced cell proliferation and induced EMT (epithelial-mesenchymal transition) phenotypes, including cell invasion. Taken together, these results define EEF1D as a critical inducer of OSCC proliferation and EMT.

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