RESUMO
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.
Assuntos
Toxinas Bacterianas/metabolismo , NAD+ Nucleosidase/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Tipo VI/química , ADP Ribose Transferases/metabolismo , Toxinas Bacterianas/química , Modelos Moleculares , NAD/metabolismo , NAD+ Nucleosidase/química , NADP/metabolismo , Fator Tu de Elongação de Peptídeos/química , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/enzimologia , Sistemas de Secreção Tipo VI/metabolismoRESUMO
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.
Assuntos
Proteínas Sanguíneas , Dopagem Esportivo , Teste em Amostras de Sangue Seco , Proteômica , Humanos , Dopagem Esportivo/prevenção & controle , Proteômica/métodos , Proteínas Sanguíneas/análise , Teste em Amostras de Sangue Seco/métodos , Teste em Amostras de Sangue Seco/normas , Masculino , Valores de Referência , Adulto , Biomarcadores/sangue , Espectrometria de Massas/métodos , Detecção do Abuso de Substâncias/métodos , Proteoma/análise , Atletas , FemininoRESUMO
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).
Assuntos
COVID-19 , Proteômica , Masculino , Feminino , Humanos , Pulmão , Capacidade Vital , Doença Crônica , LipídeosRESUMO
Structural elucidation of Gram-negative bacterial lipid A traditionally requires chemical extraction followed by tandem MS data in the negative ion mode. Previously, we reported FLAT and FLATn as methods to rapidly determine the structure of lipid A without chromatographic techniques. In this work, we extend the capability and effectiveness of these techniques to elucidate the chemical structure in a de novo manner by including the use of positive ion mode (FLAT+ and FLATn+) spectral approaches. Advantages of positive mode analysis of lipid A include the generation of more interpretable and informative fragmentation patterns that include the identification of diagnostic fragments, including selective dissociation of a glycosidic bond between two glucosamine units and the selective dissociation at the secondary acyl chain in 2'-N, allowing for the determination of the composition of fatty acids. As a proof of principle, we present here two previously uncharacterized structures of lipid A from Roseomonas mucosa (R. mucosa) and Moraxella canis (M. canis). In R. mucosa, we determined the lipid A structure with a nonconventional backbone of-ß-1,6 linked 2,3-dideoxy-2,3-diamno-d-glucopyranose further modified with galacturonic acid in the place of typical 1-phosphate, and in M. canis, we assigned a single discrete structure using the specific fragmentation patterns of terminal phosphate groups present in lipid A. Therefore, FLATn+, in combination with FLAT and FLATn, provides a multimodal structural platform for rapid structure characterization of unusual and complex lipid A structures from a single colony.
RESUMO
Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.
Assuntos
Análise de Sequência de Proteína/métodos , Imagem Individual de Molécula/métodos , Espectrometria de Massas/métodos , Nanotecnologia , Proteínas/química , Proteômica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodosRESUMO
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.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Diferenciação Celular , Fatores de Transcrição Forkhead/genética , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Estrutura Terciária de Proteína , ProteômicaRESUMO
The life of RNA polymerase II (RNAPII) transcripts is shaped by the dynamic formation of mutually exclusive ribonucleoprotein complexes (RNPs) that direct transcript biogenesis and turnover. A key regulator of RNA metabolism in the nucleus is the scaffold protein ARS2 (arsenic resistance protein 2), bound to the cap binding complex (CBC). We report here that alternative splicing of ARS2's intron 5, generates cytoplasmic isoforms that lack 270 amino acids from the N-terminal of the protein and are functionally distinct from nuclear ARS2. Switching of ARS2 isoforms within the CBC in the cytoplasm has dramatic functional consequences, changing ARS2 from a NMD inhibitor to a NMD promoter that enhances the binding of UPF1 to NCBP1 and ERF1, favouring SURF complex formation, SMG7 recruitment and transcript degradation. ARS2 isoform exchange is also relevant during arsenic stress, where cytoplasmic ARS2 promotes a global response to arsenic in a CBC-independent manner. We propose that ARS2 isoform switching promotes the proper recruitment of RNP complexes during NMD and the cellular response to arsenic stress. The existence of non-redundant ARS2 isoforms is relevant for cell homeostasis, and stress response.
Assuntos
Arsênio , Degradação do RNAm Mediada por Códon sem Sentido , Arsênio/metabolismo , Núcleo Celular/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Helicases/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
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.
Assuntos
Hemoglobinas , Proteômica , Proteômica/métodos , Hemoglobinas/análise , Hemoglobinas Glicadas , Eritrócitos/química , Espectrometria de MassasRESUMO
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.
Assuntos
Espectrometria de Mobilidade Iônica , Lipopolissacarídeos , Humanos , Lipopolissacarídeos/química , Cefotaxima , Espectrometria de Massas em Tandem , Íons/química , Escherichia coliRESUMO
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.
Assuntos
Intestinos , Células-Tronco , Acil Coenzima A , Animais , Colesterol , Feminino , Masculino , Camundongos , EsteróisRESUMO
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.
Assuntos
Cerulenina , Dinoflagellida , Policetídeo Sintases , Proliferação Nociva de Algas , Western BlottingRESUMO
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.
Assuntos
Aciltransferases/imunologia , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Lipídeo A/imunologia , Yersinia pestis/imunologia , Animais , Evolução Biológica , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único/imunologia , Células THP-1/imunologia , Células U937 , Yersinia pseudotuberculosis/imunologiaRESUMO
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.
Assuntos
COVID-19 , Biomarcadores , Humanos , Plasma , Proteômica , Estudos RetrospectivosRESUMO
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.
Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Antibacterianos/farmacologia , Colistina , Farmacorresistência Bacteriana , Escherichia coli , Infecções por Escherichia coli/tratamento farmacológico , Humanos , Lipídeo A , Testes de Sensibilidade MicrobianaRESUMO
OBJECTIVES: To determine whether angiotensin receptor blockers (ARBs) or angiotensin-converting enzyme (ACE) inhibitors are associated with improved outcomes in hospitalized patients with COVID-19 according to sex and to report sex-related differences in renin-angiotensin system (RAS) components. DESIGN: Prospective observational cohort study comparing the effects of ARB or ACE inhibitors versus no ARBs or ACE inhibitors in males versus females. Severe acute respiratory syndrome coronavirus 2 downregulates ACE-2, potentially increasing angiotensin II (a pro-inflammatory vasoconstrictor). Sex-based differences in RAS dysregulation may explain sex-based differences in responses to ARBs because the ACE2 gene is on the X chromosome. We recorded baseline characteristics, comorbidities, prehospital ARBs or ACE inhibitor treatment, use of organ support and mortality, and measured RAS components at admission and days 2, 4, 7, and 14 in a subgroup ( n = 46), recorded d -dimer ( n = 967), comparing males with females. SETTING: ARBs CORONA I is a multicenter Canadian observational cohort of patients hospitalized with acute COVID-19. This analysis includes patients admitted to 10 large urban hospitals across the four most populated provinces. PATIENTS: One-thousand six-hundred eighty-six patients with polymerase chain reaction-confirmed COVID-19 (February 2020 to March 2021) for acute COVID-19 illness were included. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Males on ARBs before admission had decreased use of ventilation (adjusted odds ratio [aOR] = 0.52; p = 0.007) and vasopressors (aOR = 0.55; p = 0.011) compared with males not on ARBs or ACE inhibitors. No significant effects were observed in females for these outcomes. The test for interaction was significant for use of ventilation ( p = 0.006) and vasopressors ( p = 0.044) indicating significantly different responses to ARBs according to sex. Males had significantly higher plasma ACE-1 at baseline and angiotensin II at day 7 and 14 than females. CONCLUSIONS: ARBs use was associated with less ventilation and vasopressors in males but not females. Sex-based differences in RAS dysregulation may contribute to sex-based differences in outcomes and responses to ARBs in COVID-19.
Assuntos
Tratamento Farmacológico da COVID-19 , Hipertensão , Angiotensina II/farmacologia , Antagonistas de Receptores de Angiotensina/farmacologia , Antagonistas de Receptores de Angiotensina/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Canadá , Feminino , Humanos , Masculino , Estudos Prospectivos , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , Caracteres SexuaisRESUMO
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.
Assuntos
Rim/química , Proteoma/análise , Formaldeído , Humanos , Espectrometria de Massas , Inclusão em Parafina , Proteômica , Fixação de TecidosRESUMO
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.
Assuntos
Proteínas , Proteômica , Sequência de Aminoácidos , Humanos , Peptídeos , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Esôfago de Barrett , Neoplasias Esofágicas , Esôfago de Barrett/genética , Linhagem Celular , Ribonucleoproteínas Nucleares Heterogêneas Grupo C , Humanos , Mucoproteínas , Proteínas Oncogênicas , Proteômica , Reprodutibilidade dos Testes , Proteínas de Ligação a Tacrolimo , Enzimas de Conjugação de UbiquitinaRESUMO
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.
Assuntos
Proteoma , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismo , Animais , Diferenciação Celular , Fatores de Transcrição Forkhead/metabolismo , Camundongos , Proteômica , Fator de Crescimento Transformador beta/metabolismo , Vimentina/metabolismoRESUMO
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.