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Urinary tract infections (UTIs) are a worldwide health problem. Fast and accurate detection of bacterial infection is essential to provide appropriate antibiotherapy to patients and to avoid the emergence of drug-resistant pathogens. While the gold standard requires 24h to 48h of bacteria culture prior MALDI-TOF species identification, we propose a culture-free workflow, enabling a bacterial identification and quantification in less than 4 hours using 1mL of urine. After a rapid and automatable sample preparation, a signature of 82 bacterial peptides, defined by machine learning, was monitored in LC-MS, to distinguish the 15 species causing 84% of the UTIs. The combination of the sensitivity of the SRM mode on a triple quadrupole TSQ Altis instrument and the robustness of capillary flow enabled us to analyze up to 75 samples per day, with 99.2% accuracy on bacterial inoculations of healthy urines. We have also shown our method can be used to quantify the spread of the infection, from 8x104 to 3x107 CFU/mL. Finally, the workflow was validated on 45 inoculated urines and on 84 UTI-positive urine from patients, with respectively 93.3% and 87.1% of agreement with the culture-MALDI procedure at a level above 1x105 CFU/mL corresponding to an infection requiring antibiotherapy.
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BACKGROUND: Emerald ash borer (Agrilus planipennis; EAB) is an Asian insect species that has been invasive to North America for 20 years. During this time, the emerald ash borer has killed tens of millions of American ash (Fraxinus spp) trees. Understanding the inherent defenses of susceptible American ash trees will provide information to breed new resistant varieties of ash trees. RESULTS: We have performed RNA-seq on naturally infested green ash (F. pennsylvanica) trees at low, medium and high levels of increasing EAB infestation and proteomics on low and high levels of EAB infestation. Most significant transcript changes we detected occurred between the comparison of medium and high levels of EAB infestation, indicating that the tree is not responding to EAB until it is highly infested. Our integrative analysis of the RNA-Seq and proteomics data identified 14 proteins and 4 transcripts that contribute most to the difference between highly infested and low infested trees. CONCLUSIONS: The putative functions of these transcripts and proteins suggests roles of phenylpropanoid biosynthesis and oxidation, chitinase activity, pectinesterase activity, strigolactone signaling, and protein turnover.
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Besouros , Fraxinus , Animais , Floema , Melhoramento Vegetal , América do Norte , ÁrvoresRESUMO
Rationale: Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by abnormally elevated pulmonary pressures and right ventricular failure. Excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) is one of the most important drivers of vascular remodeling in PAH, for which available treatments have limited effectiveness.Objectives: To gain insights into the mechanisms leading to the development of the disease and identify new actionable targets.Methods: Protein expression profiling was conducted by two-dimensional liquid chromatography coupled to tandem mass spectrometry in isolated PASMCs from controls and patients with PAH. Multiple molecular, biochemical, and pharmacologic approaches were used to decipher the role of NUDT1 (nudrix hyrolase 1) in PAH.Measurements and Main Results: Increased expression of the detoxifying DNA enzyme NUDT1 was detected in cells and tissues from patients with PAH and animal models. In vitro, molecular or pharmacological inhibition of NUDT1 in PAH-PASMCs induced accumulation of oxidized nucleotides in the DNA, irresolvable DNA damage (comet assay), disruption of cellular bioenergetics (Seahorse), and cell death (terminal deoxynucleotidyl transferase dUTP nick end labeling assay). In two animal models with established PAH (i.e., monocrotaline and Sugen/hypoxia-treated rats), pharmacological inhibition of NUDT1 using (S)-Crizotinib significantly decreased pulmonary vascular remodeling and improved hemodynamics and cardiac function.Conclusions: Our results indicate that, by overexpressing NUDT1, PAH-PASMCs hijack persistent oxidative stress in preventing incorporation of oxidized nucleotides into DNA, thus allowing the cell to escape apoptosis and proliferate. Given that NUDT1 inhibitors are under clinical investigation for cancer, they may represent a new therapeutic option for PAH.
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Enzimas Reparadoras do DNA/genética , DNA/metabolismo , Estresse Oxidativo/genética , Monoéster Fosfórico Hidrolases/genética , Hipertensão Arterial Pulmonar/genética , Artéria Pulmonar/metabolismo , Remodelação Vascular/genética , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , Adulto , Idoso , Animais , Apoptose/genética , Western Blotting , Estudos de Casos e Controles , Proliferação de Células/genética , Cromatografia Líquida , Ensaio Cometa , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismo , Modelos Animais de Doenças , Feminino , Proteína Forkhead Box M1/metabolismo , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Oxirredução , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/genética , Pirofosfatases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Espectrometria de Massas em Tandem , Regulação para CimaRESUMO
Over the past decade, the data-independent acquisition mode has gained popularity for broad coverage of complex proteomes by LC-MS/MS and quantification of low-abundance proteins. However, there is no consensus in the literature on the best data acquisition parameters and processing tools to use for this specific application. Here, we present the most comprehensive comparison of DIA workflows on Orbitrap instruments published so far in the field of proteomics. Using a standard human 48 proteins mixture (UPS1-Sigma) at 8 different concentrations in an E. coli proteome background, we tested 36 workflows including 4 different DIA window acquisition schemes and 6 different software tools (DIA-NN, DIA-Umpire, OpenSWATH, ScaffoldDIA, Skyline, and Spectronaut) with or without the use of a DDA spectral library. On the basis of the number of proteins identified, quantification linearity and reproducibility, as well as sensitivity and specificity in 28 pairwise comparisons of different UPS1 concentrations, we summarize the major considerations and propose guidelines for choosing the DIA workflow best suited for LC-MS/MS proteomic analyses. Our 96 DIA raw files and software outputs have been deposited on ProteomeXchange for testing or developing new DIA processing tools.
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Benchmarking , Proteômica , Cromatografia Líquida , Escherichia coli/genética , Humanos , Proteoma , Reprodutibilidade dos Testes , Software , Espectrometria de Massas em TandemRESUMO
Fast identification of microbial species in clinical samples is essential to provide an appropriate antibiotherapy to the patient and reduce the prescription of broad-spectrum antimicrobials leading to antibioresistances. MALDI-TOF-MS technology has become a tool of choice for microbial identification but has several drawbacks: it requires a long step of bacterial culture before analysis (≥24 h), has a low specificity and is not quantitative. We developed a new strategy for identifying bacterial species in urine using specific LC-MS/MS peptidic signatures. In the first training step, libraries of peptides are obtained on pure bacterial colonies in DDA mode, their detection in urine is then verified in DIA mode, followed by the use of machine learning classifiers (NaiveBayes, BayesNet and Hoeffding tree) to define a peptidic signature to distinguish each bacterial species from the others. Then, in the second step, this signature is monitored in unknown urine samples using targeted proteomics. This method, allowing bacterial identification in less than 4 h, has been applied to fifteen species representing 84% of all Urinary Tract Infections. More than 31,000 peptides in 190 samples were quantified by DIA and classified by machine learning to determine an 82 peptides signature and build a prediction model. This signature was validated for its use in routine using Parallel Reaction Monitoring on two different instruments. Linearity and reproducibility of the method were demonstrated as well as its accuracy on donor specimens. Within 4h and without bacterial culture, our method was able to predict the predominant bacteria infecting a sample in 97% of cases and 100% above the standard threshold. This work demonstrates the efficiency of our method for the rapid and specific identification of the bacterial species causing UTI and could be extended in the future to other biological specimens and to bacteria having specific virulence or resistance factors.
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Bactérias/classificação , Proteínas de Bactérias/urina , Bacteriúria/urina , Cromatografia Líquida/métodos , Aprendizado de Máquina , Espectrometria de Massas em Tandem/métodos , Bactérias/isolamento & purificação , Humanos , Peptídeos/urina , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The proteasome controls a multitude of cellular processes through protein degradation and has been identified as a therapeutic target in oncology. However, our understanding of its function and the development of specific modulators are hampered by the lack of a straightforward method to determine the overall proteasome status in biological samples. Here, we present a method to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes based on a robust, absolute SILAC-based multiplexed LC-Selected Reaction Monitoring (SRM) quantitative mass spectrometry assay with high precision, accuracy, and sensitivity. The method was initially optimized and validated by comparison with a reference ELISA assay and by analyzing the dynamics of catalytic subunits in HeLa cells following IFNγ-treatment and in range of human tissues. It was then successfully applied to reveal IFNγ- and O2-dependent variations of proteasome status during primary culture of Adipose-derived-mesenchymal Stromal/Stem Cells (ADSCs). The results show the critical importance of controlling the culture conditions during cell expansion for future therapeutic use in humans. We hypothesize that a shift from the standard proteasome to the immunoproteasome could serve as a predictor of immunosuppressive and differentiation capacities of ADSCs and, consequently, that quality control should include proteasomal quantification in addition to examining other essential cell parameters. The method presented also provides a new powerful tool to conduct more individualized protocols in cancer or inflammatory diseases where selective inhibition of the immunoproteasome has been shown to reduce side effects.
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Espectrometria de Massas/métodos , Células-Tronco Mesenquimais/citologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Oxigênio/farmacologia , Reprodutibilidade dos TestesRESUMO
Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by a sustained elevation of pulmonary artery (PA) pressure, right ventricular failure, and premature death. Enhanced proliferation and resistance to apoptosis (as seen in cancer cells) of PA smooth muscle cells (PASMCs) is a major pathological hallmark contributing to pulmonary vascular remodeling in PAH, for which current therapies have only limited effects. Emerging evidence points toward a critical role for Enhancer of Zeste Homolog 2 (EZH2) in cancer cell proliferation and survival. However, its role in PAH remains largely unknown. The aim of this study was to determine whether EZH2 represents a new factor critically involved in the abnormal phenotype of PAH-PASMCs. We found that EZH2 is overexpressed in human lung tissues and isolated PASMCs from PAH patients compared to controls as well as in two animal models mimicking the disease. Through loss- and gain-of-function approaches, we showed that EZH2 promotes PAH-PASMC proliferation and survival. By combining quantitative transcriptomic and proteomic approaches in PAH-PASMCs subjected or not to EZH2 knockdown, we found that inhibition of EZH2 downregulates many factors involved in cell-cycle progression, including E2F targets, and contributes to maintain energy production. Notably, we found that EZH2 promotes expression of several nuclear-encoded components of the mitochondrial translation machinery and tricarboxylic acid cycle genes. Overall, this study provides evidence that, by overexpressing EZH2, PAH-PASMCs remove the physiological breaks that normally restrain their proliferation and susceptibility to apoptosis and suggests that EZH2 or downstream factors may serve as therapeutic targets to combat pulmonary vascular remodeling.
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Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteoma/genética , Hipertensão Arterial Pulmonar/genética , Transcriptoma/genética , Animais , Apoptose/genética , Proliferação de Células/genética , Ciclo do Ácido Cítrico/genética , Epigênese Genética/genética , Feminino , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/crescimento & desenvolvimento , Artéria Pulmonar/patologia , RatosRESUMO
The production of extracellular vesicles (EV) is a ubiquitous feature of eukaryotic cells but pathological events can affect their formation and constituents. We sought to characterize the nature, profile and protein signature of EV in the plasma of Parkinson's disease (PD) patients and how they correlate to clinical measures of the disease. EV were initially collected from cohorts of PD (nâ¯=â¯60; Controls, nâ¯=â¯37) and Huntington's disease (HD) patients (Pre-manifest, nâ¯=â¯11; manifest, nâ¯=â¯52; Controls, nâ¯=â¯55) - for comparative purposes in individuals with another chronic neurodegenerative condition - and exhaustively analyzed using flow cytometry, electron microscopy and proteomics. We then collected 42 samples from an additional independent cohort of PD patients to confirm our initial results. Through a series of iterative steps, we optimized an approach for defining the EV signature in PD. We found that the number of EV derived specifically from erythrocytes segregated with UPDRS scores corresponding to different disease stages. Proteomic analysis further revealed that there is a specific signature of proteins that could reliably differentiate control subjects from mild and moderate PD patients. Taken together, we have developed/identified an EV blood-based assay that has the potential to be used as a biomarker for PD.
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Eritrócitos/metabolismo , Vesículas Extracelulares/metabolismo , Doença de Parkinson/sangue , Idoso , Biomarcadores/sangue , Contagem de Células Sanguíneas , Eritrócitos/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Feminino , Humanos , Doença de Huntington/sangue , Doença de Huntington/diagnóstico , Doença de Huntington/patologia , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/diagnóstico , Doença de Parkinson/patologia , ProteômicaRESUMO
Besides genetic abnormalities in MPN patients, several studies have reported alterations in protein expression that could contribute towards the clinical phenotype. However, little is known about protein modifications in Ph- MPN erythrocytes. In this context, we used a quantitative mass spectrometry proteomics approach to study the MPN erythrocyte proteome. LC-MS/MS (LTQ Orbitrap) analysis led to the identification of 51 and 86 overexpressed proteins in Polycythemia Vera and Essential Thrombocythemia respectively, compared with controls. Functional comparison using pathway analysis software showed that the Rho GTPase family signaling pathways were deregulated in MPN patients. In particular, IQGAP1 was significantly overexpressed in MPNs compared with controls. Additionally, Western-blot analysis not only confirmed IQGAP1 overexpression, but also showed that IQGAP1 levels depended on the patient's genotype. Moreover, we found that in JAK2V617F patients IQGAP1 could bind RhoA, Rac1 and Cdc42 and consequently recruit activated GTP-Rac1 and the cytoskeleton motility protein PAK1. In CALR(+) patients, IQGAP1 was not overexpressed but immunoprecipitated with RhoGDI. In JAK2V617F transduced Ba/F3 cells we confirmed JAK2 inhibitor-sensitive overexpression of IQGAP1/PAK1. Altogether, our data demonstrated alterations of IQGAP1/Rho GTPase signaling in MPN erythrocytes dependent on JAK2/CALR status, reinforcing the hypothesis that modifications in erythrocyte signaling pathways participate in Ph- MPN pathogenesis.
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Biomarcadores Tumorais/genética , Calreticulina/genética , Eritrócitos/enzimologia , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/enzimologia , Transdução de Sinais , Proteínas Ativadoras de ras GTPase/metabolismo , Biomarcadores Tumorais/sangue , Calreticulina/sangue , Estudos de Casos e Controles , Linhagem Celular , Cromatografia Líquida , Predisposição Genética para Doença , Humanos , Janus Quinase 2/sangue , Transtornos Mieloproliferativos/sangue , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/genética , Fenótipo , Ligação Proteica , Proteômica/métodos , Espectrometria de Massas em Tandem , Transfecção , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/sangue , Proteínas Ativadoras de ras GTPase/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Neutrophils infiltrate the airways of patients with asthma of all severities, yet their role in the pathogenesis of asthma and their contribution to airway remodeling is largely unknown. We hypothesized that neutrophils modulate airway smooth muscle (ASM) proliferation in asthma by releasing bioactive exosomes. These newly discovered nano-sized vesicles have the capacity to modulate immune responses, cell migration, cell differentiation, and other aspects of cell-to-cell communication. The aim of the study is to determine whether bioactive exosomes are released by neutrophils, and, if so, characterize their proteomic profile and evaluate their capacity to modulate ASM cell proliferation. Exosomes were isolated from equine neutrophil supernatants by differential centrifugation and filtration methods, followed by size-exclusion chromatography. Nanovesicles were characterized using electron microscopy, particle size determination, and proteomic analyses. Exosomes were cocultured with ASM cells and analyzed for exosome internalization by confocal microscopy. ASM proliferation was measured using an impedance-based system. Neutrophils release exosomes that have characteristic size, morphology, and exosomal markers. We identified 271 proteins in exosomes from both LPS and unstimulated neutrophils, and 16 proteins that were differentially expressed, which carried proteins associated with immune response and positive regulation of cell communication. Furthermore, neutrophil-derived exosomes were rapidly internalized by ASM cells and altered their proliferative properties. Upon stimulation of LPS, neutrophil-derived exosomes can enhance the proliferation of ASM cells and could therefore play an important role in the progression of asthma and promoting airway remodeling in severe and corticosteroid-insensitive patients with asthma.
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Remodelação das Vias Aéreas , Exossomos/metabolismo , Músculo Liso/fisiologia , Neutrófilos/metabolismo , Animais , Asma/patologia , Asma/fisiopatologia , Cromatografia em Gel , Cavalos , Proteoma/metabolismo , ProteômicaRESUMO
In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments.
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Proteínas Aviárias/metabolismo , Embrião de Galinha/metabolismo , Membrana Corioalantoide/metabolismo , Células Endoteliais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Animais , Biotinilação , Linhagem Celular Tumoral , Membrana Corioalantoide/lesões , Humanos , Intestino Delgado/embriologia , Intestino Delgado/metabolismo , Rim/embriologia , Rim/metabolismo , Fígado/embriologia , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , ProteomaRESUMO
Proteomic profiling provides in-depth information about the regulation of diverse biological processes, activation of and communication across signaling networks, and alterations to protein production, modifications, and interactions. For infectious disease research, mass spectrometry-based proteomics enables detection of host defenses against infection and mechanisms used by the pathogen to evade such responses. In this chapter, we outline protein extraction from organs, tissues, and fluids collected following intranasal inoculation of a murine model with the human fungal pathogen Cryptococcus neoformans. We describe sample preparation, followed by purification, processing on the mass spectrometer, and a robust bioinformatics analysis. The information gleaned from proteomic profiling of fungal infections supports the detection of novel biomarkers for diagnostic and prognostic purposes.
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Criptococose , Cryptococcus neoformans , Modelos Animais de Doenças , Proteômica , Animais , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidade , Camundongos , Criptococose/microbiologia , Criptococose/metabolismo , Proteômica/métodos , Biologia Computacional/métodos , Proteoma/metabolismo , Biomarcadores/metabolismo , Espectrometria de Massas/métodosRESUMO
Liquid Chromatography Mass Spectrometry (LC-MS) is a powerful method for profiling complex biological samples. However, batch effects typically arise from differences in sample processing protocols, experimental conditions, and data acquisition techniques, significantly impacting the interpretability of results. Correcting batch effects is crucial for the reproducibility of omics research, but current methods are not optimal for the removal of batch effects without compressing the genuine biological variation under study. We propose a suite of Batch Effect Removal Neural Networks (BERNN) to remove batch effects in large LC-MS experiments, with the goal of maximizing sample classification performance between conditions. More importantly, these models must efficiently generalize in batches not seen during training. A comparison of batch effect correction methods across five diverse datasets demonstrated that BERNN models consistently showed the strongest sample classification performance. However, the model producing the greatest classification improvements did not always perform best in terms of batch effect removal. Finally, we show that the overcorrection of batch effects resulted in the loss of some essential biological variability. These findings highlight the importance of balancing batch effect removal while preserving valuable biological diversity in large-scale LC-MS experiments.
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Espectrometria de Massa com Cromatografia Líquida , Redes Neurais de Computação , Reprodutibilidade dos TestesRESUMO
Fungal pathogens are emerging threats to global health with the rise of incidence associated with climate change and increased geographical distribution; factors also influencing host susceptibility to infection. Accurate detection and diagnosis of fungal infections is paramount to offer rapid and effective therapeutic options. For improved diagnostics, the discovery and development of protein biomarkers presents a promising avenue; however, this approach requires a priori knowledge of infection hallmarks. To uncover putative novel biomarkers of disease, profiling of the host immune response and pathogen virulence factor production is indispensable. In this study, we use mass-spectrometry-based proteomics to resolve the temporal proteome of Cryptococcus neoformans infection of the spleen following a murine model of infection. Dual perspective proteome profiling defines global remodeling of the host over a time course of infection, confirming activation of immune associated proteins in response to fungal invasion. Conversely, pathogen proteomes detect well-characterized C. neoformans virulence determinants, along with novel mapped patterns of pathogenesis during the progression of disease. Together, our innovative systematic approach confirms immune protection against fungal pathogens and explores the discovery of putative biomarker signatures from complementary biological systems to monitor the presence and progression of cryptococcal disease.
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Criptococose , Cryptococcus neoformans , Humanos , Animais , Camundongos , Proteoma , Baço/metabolismo , Criptococose/microbiologia , Criptococose/prevenção & controle , Fatores de Virulência/metabolismo , Biomarcadores , Proteínas Fúngicas/metabolismoRESUMO
Liquid Chromatography Mass Spectrometry (LC-MS) is a powerful method for profiling complex biological samples. However, batch effects typically arise from differences in sample processing protocols, experimental conditions and data acquisition techniques, significantlyimpacting the interpretability of results. Correcting batch effects is crucial for the reproducibility of proteomics research, but current methods are not optimal for removal of batch effects without compressing the genuine biological variation under study. We propose a suite of Batch Effect Removal Neural Networks (BERNN) to remove batch effects in large LC-MS experiments, with the goal of maximizing sample classification performance between conditions. More importantly, these models must efficiently generalize in batches not seen during training. Comparison of batch effect correction methods across three diverse datasets demonstrated that BERNN models consistently showed the strongest sample classification performance. However, the model producing the greatest classification improvements did not always perform best in terms of batch effect removal. Finally, we show that overcorrection of batch effects resulted in the loss of some essential biological variability. These findings highlight the importance of balancing batch effect removal while preserving valuable biological diversity in large-scale LC-MS experiments.
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Alzheimer's disease (AD) is a complex and heterogeneous neurodegenerative disorder with contributions from multiple pathophysiological pathways. One of the long-recognized and important features of AD is disrupted cerebral glucose metabolism, but the underlying molecular basis remains unclear. In this study, unbiased mass spectrometry was used to survey CSF from a large clinical cohort, comparing patients who are either cognitively unimpaired (CU; n = 68), suffering from mild-cognitive impairment or dementia from AD (MCI-AD, n = 95; DEM-AD, n = 72), or other causes (MCI-other, n = 77; DEM-other, n = 23), or Normal Pressure Hydrocephalus (NPH, n = 57). The results revealed changes related to altered glucose metabolism. In particular, two glycolytic enzymes, pyruvate kinase (PKM) and aldolase A (ALDOA), were found to be upregulated in CSF from patients with AD compared to those with other neurological conditions. Increases in full-length PKM and ALDOA levels in CSF were confirmed with immunoblotting. Levels of these enzymes furthermore correlated negatively with CSF glucose in matching CSF samples. PKM levels were also found to be increased in AD in publicly available brain-tissue data. These results indicate that ALDOA and PKM may act as technically-robust potential biomarkers of glucose metabolism dysregulation in AD.
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Doença de Alzheimer , Disfunção Cognitiva , Hidrocefalia de Pressão Normal , Humanos , Doença de Alzheimer/psicologia , Biomarcadores/líquido cefalorraquidiano , Disfunção Cognitiva/psicologia , Espectrometria de Massas , Glicólise , Glucose , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Fragmentos de Peptídeos/líquido cefalorraquidianoRESUMO
Cerebrospinal fluid (CSF) is the biological fluid in closest contact with the brain and thus contains proteins of neural cell origin. Hence, CSF is a biochemical window into the brain and is particularly attractive for the search for biomarkers of neurological diseases. However, as in the case of other biological fluids, one of the main analytical challenges in proteomic characterization of the CSF is the very wide concentration range of proteins, largely exceeding the dynamic range of current analytical approaches. Here, we used the combinatorial peptide ligand library technology (ProteoMiner) to reduce the dynamic range of protein concentration in CSF and unmask previously undetected proteins by nano-LC-MS/MS analysis on an LTQ-Orbitrap mass spectrometer. This method was first applied on a large pool of CSF from different sources with the aim to better characterize the protein content of this fluid, especially for the low abundance components. We were able to identify 1212 proteins in CSF, and among these, 745 were only detected after peptide library treatment. However, additional difficulties for clinical studies of CSF are the low protein concentration of this fluid and the low volumes typically obtained after lumbar puncture, precluding the conventional use of ProteoMiner with large volume columns for treatment of patient samples. The method has thus been optimized to be compatible with low volume samples. We could show that the treatment is still efficient with this miniaturized protocol and that the dynamic range of protein concentration is actually reduced even with small amounts of beads, leading to an increase of more than 100% of the number of identified proteins in one LC-MS/MS run. Moreover, using a dedicated bioinformatics analytical work flow, we found that the method is reproducible and applicable for label-free quantification of series of samples processed in parallel.
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Proteínas do Líquido Cefalorraquidiano/análise , Biblioteca de Peptídeos , Proteômica/métodos , Cromatografia Líquida , Humanos , Ligantes , Espectrometria de Massas , Microesferas , Neurogênese , Reprodutibilidade dos Testes , Software , Coloração e RotulagemRESUMO
Identification of bacterial species in biological samples is essential in many applications. However, the standard methods usually use a time-consuming bacterial culture (24-48 h) and sometimes lack in specificity. To overcome these limitations, we developed a new protocol, combining LC-MS/MS analysis in Data Independent Acquisition mode and machine learning algorithms, enabling the accurate identification of the bacterial species contaminating a sample in a few hours without bacterial culture. In this chapter, we describe the three steps of the protocol (spectral libraries generation, training step, identification step) to generate customized peptide signatures and use them for bacterial identification in biological samples through targeted proteomics analyses and prediction models.
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Proteômica , Espectrometria de Massas em Tandem , Bactérias/genética , Cromatografia Líquida/métodos , Aprendizado de Máquina , Peptídeos/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
In this article, we provide a proteomic reference dataset that has been initially generated for a benchmarking of software tools for Data-Independent Acquisition (DIA) analysis. This large dataset includes 96 DIA .raw files acquired from a complex proteomic standard composed of an E.coli protein background spiked-in with 8 different concentrations of 48 human proteins (UPS1 Sigma). These 8 samples were analyzed in triplicates on an Orbitrap mass spectrometer with 4 different DIA window schemes. We also provide the spectral libraries and FASTA file used for their analysis and the software outputs of the six tools used in this study: DIA-NN, Spectronaut, ScaffoldDIA, DIA-Umpire, Skyline and OpenSWATH. This dataset also contains post-processed quantification tables where the peptides and proteins have been validated, their intensities normalized and the missing values imputed with a noise value. All the files are available on ProteomeXchange. Altogether, these files represent the most comprehensive DIA reference dataset acquired on an Orbitrap instrument ever published. It will be a very useful resource to the proteomic scientists in order to assess the performance of DIA software tools or to test their processing pipelines, to the software developers to improve their tools or develop new ones and to the students for their training on proteomics data analysis.
RESUMO
BACKGROUND: Osteoarthritis (OA) is a slowly developing and debilitating disease, and there are no validated specific biomarkers for its early detection. To improve therapeutic approaches, identification of specific molecules/biomarkers enabling early determination of this disease is needed. This study aimed at identifying, with the use of proteomics/mass spectrometry, novel OA-specific serum biomarkers. As obesity is a major risk factor for OA, we discriminated obesity-regulated proteins to target only OA-specific proteins as biomarkers. METHODS: Serum from the Osteoarthritis Initiative cohort was used and divided into 3 groups: controls (n=8), OA-obese (n=10) and OA-non-obese (n=10). Proteins were identified and quantified from the liquid chromatography-tandem mass spectrometry analyses using MaxQuant software. Statistical analysis used the Limma test followed by the Benjamini-Hochberg method. To compare the proteomic profiles, the multivariate unsupervised principal component analysis (PCA) followed by the pairwise comparison was used. To select the most predictive/discriminative features, the supervised linear classification model sparse partial least squares regression discriminant analysis (sPLS-DA) was employed. Validation of three differential proteins was performed with protein-specific assays using plasma from a cohort derived from the Newfoundland Osteoarthritis. RESULTS: In total, 509 proteins were identified, and 279 proteins were quantified. PCA-pairwise differential comparisons between the 3 groups revealed that 8 proteins were differentially regulated between the OA-obese and/or OA-non-obese with controls. Further experiments using the sPLS-DA revealed two components discriminating OA from controls (component 1, 9 proteins), and OA-obese from OA-non-obese (component 2, 23 proteins). Proteins from component 2 were considered related to obesity. In component 1, compared to controls, 7 proteins were significantly upregulated by both OA groups and 2 by the OA-obese. Among upregulated proteins from both OA groups, some of them alone would not be a suitable choice as specific OA biomarkers due to their rather non-specific role or their strong link to other pathological conditions. Altogether, data revealed that the protein CRTAC1 appears to be a strong OA biomarker candidate. Other potential new biomarker candidates are the proteins FBN1, VDBP, and possibly SERPINF1. Validation experiments revealed statistical differences between controls and OA for FBN1 (p=0.044) and VDPB (p=0.022), and a trend for SERPINF1 (p=0.064). CONCLUSION: Our study suggests that 4 proteins, CRTAC1, FBN1, VDBP, and possibly SERPINF1, warrant further investigation as potential new biomarker candidates for the whole OA population.