RESUMEN
Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD(+) and NADP(+). Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.
Asunto(s)
Toxinas Bacterianas/metabolismo , NAD+ Nucleosidasa/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreción Tipo VI/química , ADP Ribosa Transferasas/metabolismo , Toxinas Bacterianas/química , Modelos Moleculares , NAD/metabolismo , NAD+ Nucleosidasa/química , NADP/metabolismo , Factor Tu de Elongación Peptídica/química , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/enzimología , Sistemas de Secreción Tipo VI/metabolismoRESUMEN
To prevent doping practices in sports, the World Anti-Doping Agency implemented the Athlete Biological Passport (ABP) program, monitoring biological variables over time to indirectly reveal the effects of doping rather than detect the doping substance or the method itself. In the context of this program, a highly multiplexed mass spectrometry-based proteomics assay for 319 peptides corresponding to 250 proteins was developed, including proteins associated with blood-doping practices. "Baseline" expression profiles of these potential biomarkers in capillary blood (dried blood spots (DBS)) were established using multiple reaction monitoring (MRM). Combining DBS microsampling with highly multiplexed MRM assays is the best-suited technology to enhance the effectiveness of the ABP program, as it represents a cost-effective and robust alternative analytical method with high specificity and selectivity of targets in the attomole range. DBS data were collected from 10 healthy athlete volunteers over a period of 140 days (28 time points per participant). These comprehensive findings provide a personalized targeted blood proteome "fingerprint" showcasing that the targeted proteome is unique to an individual and likely comparable to a DNA fingerprint. The results can serve as a baseline for future studies investigating doping-related perturbations.
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Proteínas Sanguíneas , Doping en los Deportes , Pruebas con Sangre Seca , Proteómica , Humanos , Doping en los Deportes/prevención & control , Proteómica/métodos , Proteínas Sanguíneas/análisis , Pruebas con Sangre Seca/métodos , Pruebas con Sangre Seca/normas , Masculino , Valores de Referencia , Adulto , Biomarcadores/sangre , Espectrometría de Masas/métodos , Detección de Abuso de Sustancias/métodos , Proteoma/análisis , Atletas , FemeninoRESUMEN
Many COVID-19 survivors have post-COVID-19 conditions, and females are at a higher risk. We sought to determine (1) how protein levels change from acute to post-COVID-19 conditions, (2) whether females have a plasma protein signature different from that of males, and (3) which biological pathways are associated with COVID-19 when compared to restrictive lung disease. We measured protein levels in 74 patients on the day of admission and at 3 and 6 months after diagnosis. We determined protein concentrations by multiple reaction monitoring (MRM) using a panel of 269 heavy-labeled peptides. The predicted forced vital capacity (FVC) and diffusing capacity of the lungs for carbon monoxide (DLCO) were measured by routine pulmonary function testing. Proteins associated with six key lipid-related pathways increased from admission to 3 and 6 months; conversely, proteins related to innate immune responses and vasoconstriction-related proteins decreased. Multiple biological functions were regulated differentially between females and males. Concentrations of eight proteins were associated with FVC, %, and they together had c-statistics of 0.751 (CI:0.732-0.779); similarly, concentrations of five proteins had c-statistics of 0.707 (CI:0.676-0.737) for DLCO, %. Lipid biology may drive evolution from acute to post-COVID-19 conditions, while activation of innate immunity and vascular regulation pathways decreased over that period. (ProteomeXchange identifiers: PXD041762, PXD029437).
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COVID-19 , Proteómica , Masculino , Femenino , Humanos , Pulmón , Capacidad Vital , Enfermedad Crónica , LípidosRESUMEN
The transcription factor Foxp3 is indispensible for the differentiation and function of regulatory T cells (T(reg) cells). To gain insights into the molecular mechanisms of Foxp3-mediated gene expression, we purified Foxp3 complexes and explored their composition. Biochemical and mass-spectrometric analyses revealed that Foxp3 forms multiprotein complexes of 400-800 kDa or larger and identified 361 associated proteins, â¼30% of which were transcription related. Foxp3 directly regulated expression of a large proportion of the genes encoding its cofactors. Some transcription factor partners of Foxp3 facilitated its expression. Functional analysis of the cooperation of Foxp3 with one such partner, GATA-3, provided additional evidence for a network of transcriptional regulation afforded by Foxp3 and its associates to control distinct aspects of T(reg) cell biology.
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Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Diferenciación Celular , Factores de Transcripción Forkhead/genética , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Humanos , Ratones , Ratones Transgénicos , Estructura Terciaria de Proteína , ProteómicaRESUMEN
We compared three cell isolation and two proteomic sample preparation methods for single-cell and near-single-cell analysis. Whole blood was used to quantify hemoglobin (Hb) and glycated-Hb (gly-Hb) in erythrocytes using targeted mass spectrometry and stable isotope-labeled standard peptides. Each method differed in cell isolation and sample preparation as follows: 1) FACS and automated preparation in one-pot for trace samples (autoPOTS); 2) limited dilution via microscopy and a novel rapid one-pot sample preparation method that circumvented the need for the solid-phase extraction, low-volume liquid handling instrumentation and humidified incubation chamber; and 3) CellenONE-based cell isolation and the same one-pot sample preparation method used for limited dilution. Only the CellenONE device routinely isolated single-cells from which Hb was measured to be 540-660 amol per red blood cell (RBC), which was comparable to the calculated SI reference range for mean corpuscular hemoglobin (390-540 amol/RBC). FACSAria sorter and limited dilution could routinely isolate single-digit cell numbers, to reliably quantify CMV-Hb heterogeneity. Finally, we observed that repeated measures, using 5-25 RBCs obtained from N = 10 blood donors, could be used as an alternative and more efficient strategy than single RBC analysis to measure protein heterogeneity, which revealed multimodal distribution, unique for each individual.
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Hemoglobinas , Proteómica , Proteómica/métodos , Hemoglobinas/análisis , Hemoglobina Glucada , Eritrocitos/química , Espectrometría de MasasRESUMEN
Lipopolysaccharides (LPSs) are a hallmark virulence factor of Gram-negative bacteria. They are complex, structurally heterogeneous mixtures due to variations in number, type, and position of their simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging. Here, we describe the benefits of field asymmetric ion mobility spectrometry (FAIMS) for analysis of an intact R-type lipopolysaccharide complex mixture (lipooligosaccharide; LOS). Structural characterization was performed using Escherichia coli J5 (Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine research. FAIMS gas-phase fractionation improved the (S/N) ratio and number of detected LOS species. Additionally, FAIMS allowed the separation of overlapping isobars facilitating their tandem MS characterization and unequivocal structural assignments. In addition to FAIMS gas-phase fractionation benefits, extra sorting of the structurally related LOS molecules was further accomplished using Kendrick mass defect (KMD) plots. Notably, a custom KMD base unit of [Na-H] created a highly organized KMD plot that allowed identification of interesting and novel structural differences across the different LOS ion families, i.e., ions with different acylation degrees, oligosaccharides composition, and chemical modifications. Defining the composition of a single LOS ion by tandem MS along with the organized KMD plot structural network was sufficient to deduce the composition of 181 LOS species out of 321 species present in the mixture. The combination of FAIMS and KMD plots allowed in-depth characterization of the complex LOS mixture and uncovered a wealth of novel information about its structural variations.
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Espectrometría de Movilidad Iónica , Lipopolisacáridos , Humanos , Lipopolisacáridos/química , Cefotaxima , Espectrometría de Masas en Tándem , Iones/química , Escherichia coliRESUMEN
BACKGROUND: The intestine occupies the critical interface between cholesterol absorption and excretion. Surprisingly little is known about the role of de novo cholesterol synthesis in this organ, and its relationship to whole body cholesterol homeostasis. Here, we investigate the physiological importance of this pathway through genetic deletion of the rate-limiting enzyme. METHODS: Mice lacking 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) in intestinal villus and crypt epithelial cells were generated using a Villin-Cre transgene. Plasma lipids, intestinal morphology, mevalonate pathway metabolites, and gene expression were analyzed. RESULTS: Mice with intestine-specific loss of Hmgcr were markedly smaller at birth, but gain weight at a rate similar to wild-type littermates, and are viable and fertile into adulthood. Intestine lengths and weights were greater relative to body weight in both male and female Hmgcr intestinal knockout mice. Male intestinal knockout had decreased plasma cholesterol levels, whereas fasting triglycerides were lower in both sexes. Lipidomics revealed substantial reductions in numerous nonsterol isoprenoids and sterol intermediates within the epithelial layer, but cholesterol levels were preserved. Hmgcr intestinal knockout mice also showed robust activation of SREBP-2 (sterol-regulatory element binding protein-2) target genes in the epithelium, including the LDLR (low-density lipoprotein receptor). At the cellular level, loss of Hmgcr is compensated for quickly after birth through a dramatic expansion of the stem cell compartment, which persists into adulthood. CONCLUSIONS: Loss of Hmgcr in the intestine is compatible with life through compensatory increases in intestinal absorptive surface area, LDLR expression, and expansion of the resident stem cell compartment.
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Intestinos , Células Madre , Acilcoenzima A , Animales , Colesterol , Femenino , Masculino , Ratones , EsterolesRESUMEN
Dinoflagellates are unicellular organisms that are implicated in harmful algal blooms (HABs) caused by potent toxins that are produced through polyketide synthase (PKS) pathways. However, the exact mechanisms of toxin synthesis are unknown due to a lack of genomic segregation of fat, toxins, and other PKS-based pathways. To better understand the underlying mechanisms, the actions and expression of the PKS proteins were investigated using the toxic dinoflagellate Amphidinium carterae as a model. Cerulenin, a known ketosynthase inhibitor, was shown to reduce acetate incorporation into all fat classes with the toxins amphidinol and sulpho-amphidinol. The mass spectrometry analysis of cerulenin-reacted synthetic peptides derived from ketosynthase domains of A. carterae multimodular PKS transcripts demonstrated a strong covalent bond that could be localized using collision-induced dissociation. One multi-modular PKS sequence present in all dinoflagellates surveyed to date was found to lack an AT domain in toxin-producing species, indicating trans-acting domains, and was shown by Western blotting to be post-transcriptionally processed. These results demonstrate how toxin synthesis in dinoflagellates can be differentiated from fat synthesis despite common underlying pathway.
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Cerulenina , Dinoflagelados , Sintasas Poliquetidas , Floraciones de Algas Nocivas , Western BlottingRESUMEN
Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis (Ypt) and Yersinia enterocolitica (Ye), as well as the causative agent of plague, Yersinia pestis (Yp). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye, lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2' and 3' positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp, a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion.
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Aciltransferasas/inmunología , Evasión Inmune/inmunología , Inmunidad Innata/inmunología , Lípido A/inmunología , Yersinia pestis/inmunología , Animales , Evolución Biológica , Línea Celular , Línea Celular Tumoral , Células HEK293 , Humanos , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Polimorfismo de Nucleótido Simple/inmunología , Células THP-1/inmunología , Células U937 , Yersinia pseudotuberculosis/inmunologíaRESUMEN
The host response to COVID-19 pathophysiology over the first few days of infection remains largely unclear, especially the mechanisms in the blood compartment. We report on a longitudinal proteomic analysis of acute-phase COVID-19 patients, for which we used blood plasma, multiple reaction monitoring with internal standards, and data-independent acquisition. We measured samples on admission for 49 patients, of which 21 had additional samples on days 2, 4, 7, and 14 after admission. We also measured 30 externally obtained samples from healthy individuals for comparison at baseline. The 31 proteins differentiated in abundance between acute COVID-19 patients and healthy controls belonged to acute inflammatory response, complement activation, regulation of inflammatory response, and regulation of protein activation cascade. The longitudinal analysis showed distinct profiles revealing increased levels of multiple lipid-associated functions, a rapid decrease followed by recovery for complement activation, humoral immune response, and acute inflammatory response-related proteins, and level fluctuation in the regulation of smooth muscle cell proliferation, secretory mechanisms, and platelet degranulation. Three proteins were differentiated between survivors and nonsurvivors. Finally, increased levels of fructose-bisphosphate aldolase B were determined in patients with exposure to angiotensin receptor blockers versus decreased levels in those exposed to angiotensin-converting enzyme inhibitors. Data are available via ProteomeXchange PXD029437.
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COVID-19 , Biomarcadores , Humanos , Plasma , Proteómica , Estudios RetrospectivosRESUMEN
We describe an innovative use for the recently reported fast lipid analysis technique (FLAT) that allows for the generation of MALDI tandem mass spectrometry data suitable for lipid A structure analysis directly from a single Gram-negative bacterial colony. We refer to this tandem MS version of FLAT as FLATn. Neither technique requires sophisticated sample preparation beyond the selection of a single bacterial colony, which significantly reduces overall analysis time (â¼1 h), as compared to conventional methods. Moreover, the tandem mass spectra generated by FLATn provides comprehensive information on fragments of lipid A, for example, ester bonded acyl chain dissociations, cross-ring cleavages, and glycosidic bond dissociations, all of which allow the facile determination of novel lipid A structures or confirmation of expected structures. In addition to generating tandem mass spectra directly from single colonies, we also show that FLATn can be used to analyze lipid A structures taken directly from a complex biological clinical sample without the need for ex vivo growth. From a urine sample from a patient with an E. coli infection, FLATn identified the organism and demonstrated that this clinical isolate carried the mobile colistin resistance-1 gene (mcr-1) that results in the addition of a phosphoethanolamine moiety and subsequently resistance to the antimicrobial, colistin (polymyxin E). Moreover, FLATn allowed for the determination of the existence of a structural isomer in E. coli lipid A that had either a 1- or 4'-phosphate group modification by phosphoethanolamine generated by a change of bacterial culture conditions.
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Infecciones por Escherichia coli , Proteínas de Escherichia coli , Antibacterianos/farmacología , Colistina , Farmacorresistencia Bacteriana , Escherichia coli , Infecciones por Escherichia coli/tratamiento farmacológico , Humanos , Lípido A , Pruebas de Sensibilidad MicrobianaRESUMEN
The application of proteomics to fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) human tissues is an important development spurred on by requests from stakeholder groups in clinical fields. One objective is to complement current diagnostic methods with new specific molecular information. An important goal is to achieve adequate and consistent protein recovery across and within large-scale studies. Here, we describe development of several protocols incorporating mass spectrometry compatible detergents, including Rapigest, PPS, and ProteaseMax. Methods were applied on 4 and 15 µm thick FF tissues, and 4 µm thick FFPE tissues. We evaluated sensitivity and repeatability of the methods and found that the protocol containing Rapigest enabled detection of 630 proteins from FF tissue of 1 mm2 and 15 µm thick, whereas 498 and 297 proteins were detected with the protocols containing ProteaseMax and PPS, respectively. Surprisingly, PPS-containing buffer showed good extraction of the proteins from 4 µm thick FFPE tissue with the average of 270 protein identifications (1 mm2), similar to the results on 4 µm thick FF. Moreover, we found that temperature increases during incubation with urea on 4 µm thick FF tissue revealed a decrease in the number of identified proteins and increase in the number of the carbamylated peptides.
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Riñón/química , Proteoma/análisis , Formaldehído , Humanos , Espectrometría de Masas , Adhesión en Parafina , Proteómica , Fijación del TejidoRESUMEN
Precise multiplexed quantification of proteins in biological samples can be achieved by targeted proteomics using multiple or parallel reaction monitoring (MRM/PRM). Combined with internal standards, the method achieves very good repeatability and reproducibility enabling excellent protein quantification and allowing longitudinal and cohort studies. A laborious part of performing such experiments lies in the preparation steps dedicated to the development and validation of individual protein assays. Several public repositories host information on targeted proteomics assays, including NCI's Clinical Proteomic Tumor Analysis Consortium assay portals, PeptideAtlas SRM Experiment Library, SRMAtlas, PanoramaWeb, and PeptideTracker, with all offering varying levels of details. We introduced MRMAssayDB in 2018 as an integrated resource for targeted proteomics assays. The Web-based application maps and links the assays from the repositories, includes comprehensive up-to-date protein and sequence annotations, and provides multiple visualization options on the peptide and protein level. We have extended MRMAssayDB with more assays and extensive annotations. Currently it contains >828â¯000 assays covering >51â¯000 proteins from 94 organisms, of which >17â¯000 proteins are present in >2400 biological pathways, and >48â¯000 mapping to >21â¯000 Gene Ontology terms. This is an increase of about four times the number of assays since introduction. We have expanded annotations of interaction, biological pathways, and disease associations. A newly added visualization module for coupled molecular structural annotation browsing allows the user to interactively examine peptide sequence and any known PTMs and disease mutations, and map all to available protein 3D structures. Because of its integrative approach, MRMAssayDB enables a holistic view of suitable proteotypic peptides and commonly used transitions in empirical data. Availability: http://mrmassaydb.proteincentre.com.
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Proteínas , Proteómica , Secuencia de Aminoácidos , Humanos , Péptidos , Reproducibilidad de los ResultadosRESUMEN
Moving from macroscale preparative systems in proteomics to micro- and nanotechnologies offers researchers the ability to deeply profile smaller numbers of cells that are more likely to be encountered in clinical settings. Herein a recently developed microscale proteomic method, microdroplet processing in one pot for trace samples (microPOTS), was employed to identify proteomic changes in â¼200 Barrett's esophageal cells following physiologic and radiation stress exposure. From this small population of cells, microPOTS confidently identified >1500 protein groups, and achieved a high reproducibility with a Pearson's correlation coefficient value of R > 0.9 and over 50% protein overlap from replicates. A Barrett's cell line model treated with either lithocholic acid (LCA) or X-ray had 21 (e.g., ASNS, RALY, FAM120A, UBE2M, IDH1, ESD) and 32 (e.g., GLUL, CALU, SH3BGRL3, S100A9, FKBP3, AGR2) overexpressed proteins, respectively, compared to the untreated set. These results demonstrate the ability of microPOTS to routinely identify and quantify differentially expressed proteins from limited numbers of cells.
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Esófago de Barrett , Neoplasias Esofágicas , Esófago de Barrett/genética , Línea Celular , Ribonucleoproteína Heterogénea-Nuclear Grupo C , Humanos , Mucoproteínas , Proteínas Oncogénicas , Proteómica , Reproducibilidad de los Resultados , Proteínas de Unión a Tacrolimus , Enzimas Ubiquitina-ConjugadorasRESUMEN
The transcriptional network and protein regulators that govern T helper 17 (Th17) cell differentiation have been studied extensively using advanced genomic approaches. For a better understanding of these biological processes, we have moved a step forward, from gene- to protein-level characterization of Th17 cells. Mass spectrometry-based label-free quantitative (LFQ) proteomics analysis were made of in vitro differentiated murine Th17 and induced regulatory T (iTreg) cells. More than 4,000 proteins, covering almost all subcellular compartments, were detected. Quantitative comparison of the protein expression profiles resulted in the identification of proteins specifically expressed in the Th17 and iTreg cells. Importantly, our combined analysis of proteome and gene expression data revealed protein expression changes that were not associated with changes at the transcriptional level. Our dataset provides a valuable resource, with new insights into the proteomic characteristics of Th17 and iTreg cells, which may prove useful in developing treatment of autoimmune diseases and developing tumor immunotherapy.
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Proteoma , Linfocitos T Reguladores/metabolismo , Células Th17/metabolismo , Animales , Diferenciación Celular , Factores de Transcripción Forkhead/metabolismo , Ratones , Proteómica , Factor de Crecimiento Transformador beta/metabolismo , Vimentina/metabolismoRESUMEN
Sepsis remains one of the most lethal and costly conditions treated in U.S. hospitals, with approximately 50% of cases caused by Gram-negative bacterial infections. Septic shock is induced when lipopolysaccharide (LPS), the main component of Gram-negative outer bacterial membrane, signals through the Toll-like receptor 4 (TLR4) complex. Lethal endotoxemia, a model for septic shock, was induced in WT C57BL6 and TLR4-/- mice by administration of Escherichia coli LPS. WT LPS treated mice showed high morbidity, while PBS treated LPS and treated TLR4-/- mice did not. ANOVA analysis of label-free quantification of longitudinal serum proteome revealed 182 out of 324 proteins in LPS injected WT mice that were significantly changed across four time points (0, 6, 12, and 18 h). No significant changes were identified in the two control groups. From the 182 identified proteins, examples of known sepsis biomarkers were validated by ELISA, which showed similar trends as MS proteomics data. Longitudinal analysis within individual mice produced 3-fold more significantly changed proteins than pair-wise comparison. A subsequent global analysis of WT and TLR4-/- mice identified pathways activated independent of TLR4. These pathways represent possible compensatory mechanisms that allow for control of Gram-negative bacterial infection regardless of host immune status.
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Sepsis , Choque Séptico , Animales , Lipopolisacáridos/toxicidad , Ratones , Proteómica , Sepsis/genética , Receptor Toll-Like 4/genéticaRESUMEN
We developed a method to directly detect and map the Gram-negative bacterial virulence factor lipid A derived from lipopolysaccharide (LPS) by coupling acid hydrolysis with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). As the structure of lipid A (endotoxin) determines the innate immune outcome during infection, the ability to map its location within an infected organ or animal is needed to understand localized inflammatory responses that results during host-pathogen interactions. We previously demonstrated detection of free lipid A from infected tissue; however detection of lipid A derived from intact (smooth) LPS from host-pathogen MSI studies, proved elusive. Here, we detected LPS-derived lipid A from the Gram-negative pathogens, Escherichia coli (Ec, m/z 1797) and Pseudomonas aeruginosa (Pa, m/z 1446) using on-tissue acid hydrolysis to cleave the glycosidic linkage between the polysaccharide (core and O-antigen) and lipid A moieties of LPS. Using accurate mass methods, the ion corresponding to the major Ec and Pa lipid A species (m/z 1797 and 1446, respectively) were unambiguously discriminated from complex tissue substrates. Further, we evaluated potential delocalization and signal loss of other tissue lipids and found no evidence for either, making this LPS-to-Lipid A-MSI (LLA-MSI) method, compatible with simultaneous host-pathogen lipid imaging following acid hydrolysis. This spatially sensitive technique is the first step in mapping host-influenced de novo lipid A modifications, such as those associated with antimicrobial resistance phenotypes, during Gram-negative bacterial infection and will advance our understanding of the host-pathogen interface.
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Lípido A/análisis , Lipopolisacáridos/metabolismo , Animales , Escherichia coli/metabolismo , Riñón/microbiología , Límite de Detección , Ratones , Pseudomonas aeruginosa/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Tissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues, among which the most popular are fresh-frozen and formalin-fixed paraffin embedded methods. Depending on the preservation method and the amount of sample available, various specific protocols are available for tissue processing for subsequent proteomic analysis. Protocols are tailored to answer various biological questions, and as such vary in lysis and digestion conditions, as well as duration. The existence of diverse tissue-sample protocols has led to confusion in how to choose the best protocol for a given tissue and made it difficult to compare results across sample types. Here, we summarize procedures used for tissue processing for subsequent bottom-up proteomic analysis. Furthermore, we compare protocols for their variations in the composition of lysis buffers, digestion procedures, and purification steps. For example, reports have shown that lysis buffer composition plays an important role in the profile of extracted proteins: the most common are tris(hydroxymethyl)aminomethane, radioimmunoprecipitation assay, and ammonium bicarbonate buffers. Although, trypsin is the most commonly used enzyme for proteolysis, in some protocols it is supplemented with Lys-C and/or chymotrypsin, which will often lead to an increase in proteome coverage. Data show that the selection of the lysis procedure might need to be tissue-specific to produce distinct protocols for individual tissue types. Finally, selection of the procedures is also influenced by the amount of sample available, which range from biopsies or the size of a few dozen of mm2 obtained with laser capture microdissection to much larger amounts that weight several milligrams.
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Espectrometría de Masas/métodos , Proteoma/análisis , Proteómica/métodos , Animales , Biopsia , Cromatografía Liquida/métodos , Humanos , Proteínas/análisis , Proteolisis , Conservación de Tejido/métodosRESUMEN
RATIONALE: Polymicrobial samples present unique challenges for mass spectrometric identification. A recently developed glycolipid technology has the potential to accurately identify individual bacterial species from polymicrobial samples. In order to develop and validate bacterial identification algorithms (e.g. machine learning) using this glycolipid technology, generating a large number of various polymicrobial samples can be beneficial, but it is costly and labor-intensive. Here, we propose an alternative cost-effective approach that generates realistic in silico polymicrobial glycolipid mass spectra. METHODS: We introduce MGMS2 (membrane glycolipid mass spectrum simulator) as a simulation software package that generates in silico polymicrobial membrane glycolipid matrix-assisted laser desorption/ionization time-of-flight mass spectra. Unlike currently available simulation algorithms for polymicrobial mass spectra, the proposed algorithm considers errors in m/z values and variances of intensity values, occasions of missing signature ions, and noise peaks. To our knowledge, this is the first stand-alone bacterial membrane glycolipid mass spectral simulator. MGMS2 software and its manual are freely available as an R package. An interactive MGSM2 app that helps users explore various simulation parameter options is also available. RESULTS: We demonstrated the performance of MGSM2 using six microbes. The software generated in silico glycolipid mass spectra that are similar to real polymicrobial glycolipid mass spectra. The maximum correlation between in silico mass spectra generated by MGMS2 and the real polymicrobial mass spectrum was about 87%. CONCLUSIONS: We anticipate that MGMS2, which considers spectrum-to-spectrum variation, will advance the bacterial algorithm development for polymicrobial samples.
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Técnicas Bacteriológicas/métodos , Glucolípidos/análisis , Lípidos de la Membrana/análisis , Programas Informáticos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Bacterias/química , Biología Computacional/métodos , Simulación por Computador , Glucolípidos/química , Lípidos de la Membrana/química , Flujo de TrabajoRESUMEN
By circumventing the need for a pure colony, MALDI-TOF mass spectrometry of bacterial membrane glycolipids (lipid A) has the potential to identify microbes more rapidly than protein-based methods. However, currently available bioinformatics algorithms (e.g., dot products) do not work well with glycolipid mass spectra such as those produced by lipid A, the membrane anchor of lipopolysaccharide. To address this issue, we propose a spectral library approach coupled with a machine learning technique to more accurately identify microbes. Here, we demonstrate the performance of the model-based spectral library approach for microbial identification using approximately a thousand mass spectra collected from multi-drug-resistant bacteria. At false discovery rates < 1%, our approach identified many more bacterial species than the existing approaches such as the Bruker Biotyper and characterized over 97% of their phenotypes accurately. As the diversity in our glycolipid mass spectral library increases, we anticipate that it will provide valuable information to more rapidly treat infected patients.