Proteomics reveals disturbances in the immune response and energy metabolism of monocytes from patients with septic shock.

Sepsis results from a dyshomeostatic response to infection, which may lead to hyper or hypoimmune states. Monocytes are central regulators of the inflammatory response, but our understanding of their role in the genesis and resolution of sepsis is still limited. Here, we report a comprehensive exploration of monocyte molecular responses in a cohort of patients with septic shock via proteomic profiling. The acute stage of septic shock was associated with an impaired inflammatory phenotype, indicated by the down-regulation of MHC class II molecules and proinflammatory cytokine pathways. Simultaneously, there was an up-regulation of glycolysis enzymes and a decrease in proteins related to the citric acid cycle and oxidative phosphorylation. On the other hand, the restoration of immunocompetence was the hallmark of recovering patients, in which an upregulation of interferon signaling pathways was a notable feature. Our results provide insights into the immunopathology of sepsis and propose that, pending future studies, immunometabolism pathway components could serve as therapeutic targets in septic patients.

Glycolytic profile shift and antioxidant triggering in symbiont-free and H2O2-resistant Strigomonas culicis.

During their life cycle, trypanosomatids are exposed to stress conditions and adapt their energy and antioxidant metabolism to colonize their hosts. Strigomonas culicis is a monoxenous protist found in invertebrates with an endosymbiotic bacterium that completes essential biosynthetic pathways for the trypanosomatid. Our research group previously generated a wild-type H2O2-resistant (WTR) strain that showed improved mitochondrial metabolism and antioxidant defenses, which led to higher rates of Aedes aegypti infection. Here, we assess the biological contribution of the S. culicis endosymbiont and reactive oxygen species (ROS) resistance to oxidative and energy metabolism processes. Using high-throughput proteomics, several proteins involved in glycolysis and gluconeogenesis, the pentose phosphate pathway and glutathione metabolism were identified. The results suggest that ROS resistance decreases glucose consumption and indicate that the metabolic products from gluconeogenesis are key to supplying the protist with high-energy and reducing intermediates. Our hypothesis was confirmed by biochemical assays showing opposite profiles for glucose uptake and hexokinase and pyruvate kinase activity levels in the WTR and aposymbiotic strains, while the enzyme glucose-6P 1-dehydrogenase was more active in both strains. Regarding the antioxidant system, ascorbate peroxidase has an important role in H2O2 resistance and may be responsible for the high infection rates previously described for A. aegypti. In conclusion, our data indicate that the energy-related and antioxidant metabolic processes of S. culicis are modulated in response to oxidative stress conditions, providing new perspectives on the biology of the trypanosomatid-insect interaction as well as on the possible impact of resistant parasites in accidental human infection.

Mixed-data acquisition: next-generation quantitative proteomics data acquisition

We present the Mixed-Data Acquisition (MDA) strategy for mass spectrometry data acquisition. MDA combines Data-Dependent Acquisition (DDA) and Data-Independent Acquisition (DIA) in the same run, thus doing away with the requirements for separate DDA spectral libraries. MDA is a natural result from advances in mass spectrometry, such as high scan rates and multiple analyzers, and is tailored toward exploiting these features. We demonstrate MDA's effectiveness on a yeast proteome analysis by overcoming a common bottleneck for XIC-based label-free quantitation; namely, the coelution of precursors when m/z values cannot be distinguished. We anticipate that MDA will become the next mainstream data generation approach for proteomics. MDA can also serve as an orthogonal validation approach for DDA experiments. Specialized software for MDA data analysis is made available on the project's website.

Differential proteomic comparison of breast cancer secretome using a quantitative paired analysis workflow.

BACKGROUND: Worldwide, breast cancer is the main cause of cancer mortality in women. Most cases originate in mammary ductal cells that produce the nipple aspirate fluid (NAF). In cancer patients, this secretome contains proteins associated with the tumor microenvironment. NAF studies are challenging because of inter-individual variability. We introduced a paired-proteomic shotgun strategy that relies on NAF analysis from both breasts of patients with unilateral breast cancer and extended PatternLab for Proteomics software to take advantage of this setup. METHODS: The software is based on a peptide-centric approach and uses the binomial distribution to attribute a probability for each peptide as being linked to the disease; these probabilities are propagated to a final protein p-value according to the Stouffer's Z-score method. RESULTS: A total of 1227 proteins were identified and quantified, of which 87 were differentially abundant, being mainly involved in glycolysis (Warburg effect) and immune system activation (activated stroma). Additionally, in the estrogen receptor-positive subgroup, proteins related to the regulation of insulin-like growth factor transport and platelet degranulation displayed higher abundance, confirming the presence of a proliferative microenvironment. CONCLUSIONS: We debuted a differential bioinformatics workflow for the proteomic analysis of NAF, validating this secretome as a treasure-trove for studying a paired-organ cancer type.

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses.

Differential Gel Electrophoresis (DIGE) Evaluation of Naphthoimidazoles Mode of Action: A Study in Trypanosoma cruzi Bloodstream Trypomastigotes.

BACKGROUND: The obligate intracellular protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a neglected illness affecting millions of people in Latin America that recently entered non-endemic countries through immigration, as a consequence of globalization. The chemotherapy for this disease is based mainly on benznidazole and nifurtimox, which are very efficient nitroderivatives against the acute stage but present limited efficacy during the chronic phase. Our group has been studying the trypanocidal effects of naturally occurring quinones and their derivatives, and naphthoimidazoles derived from ß-lapachone N1, N2 and N3 were the most active. To assess the molecular mechanisms of action of these compounds, we applied proteomic techniques to analyze treated bloodstream trypomastigotes, which are the clinically relevant stage of the parasite. METHODOLOGY/PRINCIPAL FINDINGS: The approach consisted of quantification by 2D-DIGE followed by MALDI-TOF/TOF protein identification. A total of 61 differentially abundant protein spots were detected when comparing the control with each N1, N2 or N3 treatment, for 34 identified spots. Among the differentially abundant proteins were activated protein kinase C receptor, tubulin isoforms, asparagine synthetase, arginine kinase, elongation factor 2, enolase, guanine deaminase, heat shock proteins, hypothetical proteins, paraflagellar rod components, RAB GDP dissociation inhibitor, succinyl-CoA ligase, ATP synthase subunit B and methionine sulfoxide reductase. CONCLUSION/SIGNIFICANCE: Our results point to different modes of action for N1, N2 and N3, which indicate a great variety of metabolic pathways involved and allow for novel perspectives on the development of trypanocidal agents.

Proteomic profiling of nipple aspirate fluid (NAF): Exploring the complementarity of different peptide fractionation strategies.

NAF is a breast fluid that is closely related to the tumor microenvironment and a valuable sample for studying breast cancer. To perform an in-depth proteomic analysis of this sample, aliquots of a single NAF digest were analyzed by the following peptide-centric fractionation strategies: a) 30-cm reversed-phase (RP) column on-line with an LTQ-Orbitrap XL; b) off-line strong cation-exchange (SCX) column; and c) pI-based OFFGEL fractionation. All fractions from approaches (b) and (c) were further analyzed on a 10-cm RP column hyphenated to the same mass spectrometer. The RP-30cm, SCX/RP-10cm, and OFFGEL/RP-10cm approaches identified 1676, 2930, and 3240 peptides, which corresponded to 193, 390 and 528 proteins, respectively. In our cumulative dataset, 4466 distinct NAF peptides corresponded to a total of 557 proteins, of which only 34% were identified by all three approaches. No exclusive protein identification was associated to the RP-30cm approach, while SCX/RP-10cm and OFFGEL/RP-10cm contributed to 28 and 166 exclusive identifications, respectively. Each approach provided additional information related to energy metabolism (fermentation process/carbohydrate biosynthesis). In conclusion, the pre-fractionation platforms used were complementary for the comprehensive characterization of NAF and our work provides methodological information for future quantitative cancer-related NAF sample studies. BIOLOGICAL SIGNIFICANCE: High-resolution peptide separation is a sine qua non condition for achieving extensive proteome coverage. Various techniques have been employed to improve peptide fractionation prior to LC-MS/MS, thus allowing a comprehensive characterization of complex biological samples. Although fractionation efficiency is very sample-dependent, this issue is commonly overlooked, and a "cookbook" approach is routinely used during this critical step. The present study provides a systematic comparison of analytical information needed for the successful large-scale differential proteomic analysis of NAF samples from breast cancer patients, a precious and volume-limited biological sample. It reinforces the importance of optimizing sample-specific fractionation protocols for information retrieval from mass spectrometric analysis.

Reevaluating the Trypanosoma cruzi proteomic map: The shotgun description of bloodstream trypomastigotes.

Chagas disease is a neglected disease, caused by the protozoan Trypanosoma cruzi. This kinetoplastid presents a cycle involving different forms and hosts, being trypomastigotes the main infective form. Despite various T. cruzi proteomic studies, the assessment of bloodstream trypomastigote profile remains unexplored. The aim of this work is T. cruzi bloodstream form proteomic description. Employing shotgun approach, 17,394 peptides were identified, corresponding to 7514 proteins of which 5901 belong to T. cruzi. Cytoskeletal proteins, chaperones, bioenergetics-related enzymes, and trans-sialidases are among the top-scoring. GO analysis revealed that all T. cruzi compartments were assessed; and majority of proteins are involved in metabolic processes and/or presented catalytic activity. The comparative analysis between the bloodstream trypomastigotes and cultured-derived or metacyclic trypomastigote proteomic profiles pointed to 2202 proteins exclusively detected in the bloodstream form. These exclusive proteins are related to: (a) surface proteins; (b) non-classical secretion pathway; (c) cytoskeletal dynamics; (d) cell cycle and transcription; (e) proteolysis; (f) redox metabolism; (g) biosynthetic pathways; (h) bioenergetics; (i) protein folding; (j) cell signaling; (k) vesicular traffic; (l) DNA repair; and (m) cell death. This large-scale evaluation of bloodstream trypomastigotes, responsible for the parasite dissemination in the patient, marks a step forward in the comprehension of Chagas disease pathogenesis. BIOLOGICAL SIGNIFICANCE: The hemoflagellate protozoan T. cruzi is the etiological agent of Chagas disease and affects people by the millions in Latin America and other non-endemic countries. The absence of efficient drugs, especially for treatment during the chronic phase of the disease, stimulates the continuous search for novel molecular targets. The identification of essential molecules, particularly those found in clinically relevant forms of the parasite, could be crucial. Inside the vertebrate host, trypomastigotes circulate in the bloodstream before infecting various tissues. The exposure of bloodstream forms of the parasite to the host immune system likely leads to differential protein expression in the parasite. In this context, an extensive characterization of the proteomic profile of bloodstream trypomastigotes could help to find not only promising drug targets but also antigens for vaccines or diagnostics. This work is a large-scale proteomic assessment of bloodstream trypomastigotes that show a considerable number of proteins belonging to different metabolic pathways and functions exclusive to this parasitic form, and provides a valuable dataset for the biological understanding of this clinically relevant form of T. cruzi.

Potential correlation between tumor aggressiveness and protein expression patterns of nipple aspirate fluid (NAF) revealed by gel-based proteomic analysis.

Breast cancer is the leading cause of cancer related deaths in women. Most breast cancers stem from mammary ductal cells that secrete nipple aspirate fluid (NAF), a biological sample that contains proteins associated with the tumor microenvironment. In this study, NAF samples from both breasts of 7 Brazilian patients with unilateral breast cancer were analyzed. These samples were systematically compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional fluorescence difference gel electrophoresis (2D-DIGE); substantial qualitative individual differences were observed. In general, when NAF samples were compared from both breasts within the same patient their electrophoretic patterns were very similar, regardless of their cancer status. A comparison of all patients identified 2 main NAF protein profiles. The HomEP, homogeneous expression profile, was characterized by typical SDS-PAGE and 2D-DIGE protein patterns that were observed in patients with a good breast cancer prognosis and were similar to previous Type I NAF classifications that used one-dimensional electrophoresis. The HetEP, heterogeneous expression profile, was characterized by distinct protein patterns that have not been reported in previous studies and have been primarily observed in breast cancer patients with a poor prognosis. The NAF samples were rich in metal-dependent proteolytic enzymes, as visualized by SDS-PAGE zymography. They varied qualitatively with respect to their gelatinolytic band distribution. However, there were no correlations between these characteristics and the pathologic features of these tumors. A comparative analysis of NAF samples taken from each breast in a single patient showed conserved zymographic patterns. In conclusion, the present study highlights important distinctions in the protein content of individual NAF samples and provides insight into the composition of the tumor microenvironment. These data reinforce breast cancer as a heterogeneous disease with a diverse natural history, which is becoming increasingly evident through other recent studies.

Caracterização proteica do fluido aspirado de mama de pacientes brasileiras com câncer de mama unilateral / Protein characterization of breast fluid aspirated from Brazilian patients with unilateral breast cancer

O câncer de mama é a principal causa de morte por câncer em mulheres no mundo. De acordo com o Instituto Nacional do Câncer, 7.470 novos casos são estimados em 2010 apenas no Rio de Janeiro. A maioria dos casos de câncer de mama são originados nas células ductais mamárias. Elas secretam o fluido aspirado de mama (NAF), que contém proteínas intimamente associadas ao microambiente tumoral. O objetivo deste trabalho foi utilizar a abordagem proteômica como ferramenta para caracterizar e comparar o NAF das duas mamas de pacientes brasileiras com câncer da mama unilateral. A concentração média de proteínas das amostras controle, tumor benigno ou câncer de mama de 13 mulheres foi aproximadamente 115 mais ou menos 60 (miu)g/(miu)L. A atividade proteolítica do NAF foi visualizada porzimografia das amostras de duas pacientes. Observamos que o padrão de bandas gelatinolíticas diferiu substancialmente entre as pacientes, mas não entre as duas mamas do mesmo indivíduo. As enzimas proteolíticas se apresentam sensíveis à inibição por agentes quelantes de metais (EDTA e orto-fenantrolina). As amostras de NAF também foram submetidas à 2D-PAGE em condições otimizadas (IPG 4-7 e aplicação de amostra por reidratação in gel) e poucos spots protéicos muito abundantes foram detectados e identificados por espectrometria de massas MALDI-TOF/TOF como albumina, imunoglobinas, zinco-alfa2-glicoproteína e proteína induzida por prolactina. Para enriquecer o NAF em suas proteínas menos abundantes, testamos estratégias de depleção usando a coluna de múltipla afinidade Hu-6 da Agilent ou proteína L agarose. Este passo de pré-fracionamento introduziu variabilidade adicional importante nas amostras e por este motivo não foi empregado em etapas posteriores do estudo. Catorze amostras de NAF das mamas pareadas de mulheres com câncer unilateral foram sistematicamente comparadas por 2D-DIGE. Foram observadas diferenças qualitativas substanciais entre as pacientes, prejudicando as comparações quantitativas entre as amostras. Dois grandes perfis protéicos distintos foram definidos por 2D-DIGE. O perfil A caracterizou-se pela presença de dois grupos de spots ácidos típicos (pl 4,5 e MM 29,5 e 18 kDa), ausentes no perfil B. A presença de glico- e fosfo-proteínas foi determinada em amostras de ambos os perfis após revelação dos géis 2D com ProQ-Emerald ou Diamond, respectivamente. As amostras dos dois perfis se mostraram muito mais ricas em glicoproteínas do que em fosfoproteínas. Este trabalho constituiu a primeira caracterização proteômica sistemática do NAF de pacientes brasileiras com câncer de mama unilateral. Nossos resultados mostram uma variabilidade importante do conteúdo de proteínas nas amostras individuais de NAF, sugerindo a necessidade de uma estratificação mais uniforme das pacientes. Por fim, o emprego de tecnologias proteômicas mais sensíveis e acuradas deverá ser necessário para possibilitar a detecção e identificação de proteínas menos abundantes no NAF, que poderão constituir candidatos a biomarcardores do câncer de mama.