RESUMEN
MOTIVATION: The proteomics field requires the production and publication of reliable mass spectrometry-based identification and quantification results. Although many tools or algorithms exist, very few consider the importance of combining, in a unique software environment, efficient processing algorithms and a data management system to process and curate hundreds of datasets associated with a single proteomics study. RESULTS: Here, we present Proline, a robust software suite for analysis of MS-based proteomics data, which collects, processes and allows visualization and publication of proteomics datasets. We illustrate its ease of use for various steps in the validation and quantification workflow, its data curation capabilities and its computational efficiency. The DDA label-free quantification workflow efficiency was assessed by comparing results obtained with Proline to those obtained with a widely used software using a spiked-in sample. This assessment demonstrated Proline's ability to provide high quantification accuracy in a user-friendly interface for datasets of any size. AVAILABILITY AND IMPLEMENTATION: Proline is available for Windows and Linux under CECILL open-source license. It can be deployed in client-server mode or in standalone mode at http://proline.profiproteomics.fr/#downloads. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Prolina , Proteómica , Algoritmos , Espectrometría de Masas , Programas InformáticosRESUMEN
T-cell receptor (TCR) ligation-mediated protein phosphorylation regulates the activation, cellular responses, and fates of T cells. Here, we used time-resolved high-resolution phosphoproteomics to identify, quantify, and characterize the phosphorylation dynamics of thousands of phosphorylation sites in primary T cells during the first 10 min after TCR stimulation. Bioinformatic analysis of the data revealed a coherent orchestration of biological processes underlying T-cell activation. In particular, functional modules associated with cytoskeletal remodeling, transcription, translation, and metabolic processes were mobilized within seconds after TCR engagement. Among proteins whose phosphorylation was regulated by TCR stimulation, we demonstrated, using a fast-track gene inactivation approach in primary lymphocytes, that the ITSN2 adaptor protein regulated T-cell effector functions. This resource, called LymphoAtlas, represents an integrated pipeline to further decipher the organization of the signaling network encoding T-cell activation. LymphoAtlas is accessible to the community at: https://bmm-lab.github.io/LymphoAtlas.
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Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismo , Proteómica , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/genética , Animales , Anticuerpos/farmacología , Linfocitos T CD4-Positivos/inmunología , Cromatografía Liquida , Biología Computacional , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Fosforilación , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/inmunología , Transducción de Señal/inmunología , Espectrometría de Masas en Tándem , Factores de TiempoRESUMEN
Acute liver injury (ALI) is a severe disorder resulting from excessive hepatocyte cell death, and frequently caused by acetaminophen intoxication. Clinical management of ALI progression is hampered by the dearth of blood biomarkers available. In this study, a bioinformatics workflow was developed to screen omics databases and identify potential biomarkers for hepatocyte cell death. Then, discovery proteomics was harnessed to select from among these candidates those that were specifically detected in the blood of acetaminophen-induced ALI patients. Among these candidates, the isoenzyme alcohol dehydrogenase 1B (ADH1B) was massively leaked into the blood. To evaluate ADH1B, we developed a targeted proteomics assay and quantified ADH1B in serum samples collected at different times from 17 patients admitted for acetaminophen-induced ALI. Serum ADH1B concentrations increased markedly during the acute phase of the disease, and dropped to undetectable levels during recovery. In contrast to alanine aminotransferase activity, the rapid drop in circulating ADH1B concentrations was followed by an improvement in the international normalized ratio (INR) within 10-48 h, and was associated with favorable outcomes. In conclusion, the combination of omics data exploration and proteomics revealed ADH1B as a new blood biomarker candidate that could be useful for the monitoring of acetaminophen-induced ALI.
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Alcohol Deshidrogenasa/sangre , Biomarcadores/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Proteómica/métodos , Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Cromatografía Líquida de Alta Presión , Biología Computacional , Humanos , Relación Normalizada Internacional , Límite de Detección , Espectrometría de Masas en TándemRESUMEN
Urothelial bladder cancer is a condition associated with high recurrence and substantial morbidity and mortality. Noninvasive urinary tests that would detect bladder cancer and tumor recurrence are required to significantly improve patient care. Over the past decade, numerous bladder cancer candidate biomarkers have been identified in the context of extensive proteomics or transcriptomics studies. To translate these findings in clinically useful biomarkers, the systematic evaluation of these candidates remains the bottleneck. Such evaluation involves large-scale quantitative LC-SRM (liquid chromatography-selected reaction monitoring) measurements, targeting hundreds of signature peptides by monitoring thousands of transitions in a single analysis. The design of highly multiplexed SRM analyses is driven by several factors: throughput, robustness, selectivity and sensitivity. Because of the complexity of the samples to be analyzed, some measurements (transitions) can be interfered by coeluting isobaric species resulting in biased or inconsistent estimated peptide/protein levels. Thus the assessment of the quality of SRM data is critical to allow flagging these inconsistent data. We describe an efficient and robust method to process large SRM data sets, including the processing of the raw data, the detection of low-quality measurements, the normalization of the signals for each protein, and the estimation of protein levels. Using this methodology, a variety of proteins previously associated with bladder cancer have been assessed through the analysis of urine samples from a large cohort of cancer patients and corresponding controls in an effort to establish a priority list of most promising candidates to guide subsequent clinical validation studies.
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Biomarcadores de Tumor/orina , Carcinoma de Células Transicionales/orina , Proteómica , Neoplasias de la Vejiga Urinaria/orina , Secuencia de Aminoácidos/genética , Biomarcadores de Tumor/genética , Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/patología , Cromatografía Liquida/métodos , Humanos , Espectrometría de Masas/métodos , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
The Chromosome-Centric Human Proteome Project (C-HPP) aims to identify "missing" proteins in the neXtProt knowledgebase. We present an in-depth proteomics analysis of the human sperm proteome to identify testis-enriched missing proteins. Using protein extraction procedures and LC-MS/MS analysis, we detected 235 proteins (PE2-PE4) for which no previous evidence of protein expression was annotated. Through LC-MS/MS and LC-PRM analysis, data mining, and immunohistochemistry, we confirmed the expression of 206 missing proteins (PE2-PE4) in line with current HPP guidelines (version 2.0). Parallel reaction monitoring acquisition and sythetic heavy labeled peptides targeted 36 âªone-hit wonderâ« candidates selected based on prior peptide spectrum match assessment. 24 were validated with additional predicted and specifically targeted peptides. Evidence was found for 16 more missing proteins using immunohistochemistry on human testis sections. The expression pattern for some of these proteins was specific to the testis, and they could possibly be valuable markers with fertility assessment applications. Strong evidence was also found of four "uncertain" proteins (PE5); their status should be re-examined. We show how using a range of sample preparation techniques combined with MS-based analysis, expert knowledge, and complementary antibody-based techniques can produce data of interest to the community. All MS/MS data are available via ProteomeXchange under identifier PXD003947. In addition to contributing to the C-HPP, we hope these data will stimulate continued exploration of the sperm proteome.
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Proteoma/análisis , Espermatozoides/química , Cromatografía Liquida , Minería de Datos , Bases de Datos de Proteínas , Humanos , Inmunohistoquímica , Masculino , Proteómica/métodos , Espectrometría de Masas en Tándem , Testículo/químicaRESUMEN
Metabolic engineering aims to design high performance microbial strains producing compounds of interest. This requires systems-level understanding; genome-scale models have therefore been developed to predict metabolic fluxes. However, multi-omics data including genomics, transcriptomics, fluxomics, and proteomics may be required to model the metabolism of potential cell factories. Recent technological advances to quantitative proteomics have made mass spectrometry-based quantitative assays an interesting alternative to more traditional immuno-affinity based approaches. This has improved specificity and multiplexing capabilities. In this study, we developed a quantification workflow to analyze enzymes involved in central metabolism in Escherichia coli (E. coli). This workflow combined full-length isotopically labeled standards with selected reaction monitoring analysis. First, full-length (15)N labeled standards were produced and calibrated to ensure accurate measurements. Liquid chromatography conditions were then optimized for reproducibility and multiplexing capabilities over a single 30-min liquid chromatography-MS analysis. This workflow was used to accurately quantify 22 enzymes involved in E. coli central metabolism in a wild-type reference strain and two derived strains, optimized for higher NADPH production. In combination with measurements of metabolic fluxes, proteomics data can be used to assess different levels of regulation, in particular enzyme abundance and catalytic rate. This provides information that can be used to design specific strains used in biotechnology. In addition, accurate measurement of absolute enzyme concentrations is key to the development of predictive kinetic models in the context of metabolic engineering.
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Carbono/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Espectrometría de Masas/métodos , NADP/metabolismo , Calibración , Cromatografía Liquida/métodos , Marcaje Isotópico , Cinética , Ingeniería Metabólica , Proteómica/métodos , Estándares de Referencia , Reproducibilidad de los Resultados , Flujo de TrabajoRESUMEN
In the framework of the C-HPP, our Franco-Swiss consortium has adopted chromosomes 2 and 14, coding for a total of 382 missing proteins (proteins for which evidence is lacking at protein level). Over the last 4 years, the French proteomics infrastructure has collected high-quality data sets from 40 human samples, including a series of rarely studied cell lines, tissue types, and sample preparations. Here we described a step-by-step strategy based on the use of bioinformatics screening and subsequent mass spectrometry (MS)-based validation to identify what were up to now missing proteins in these data sets. Screening database search results (85,326 dat files) identified 58 of the missing proteins (36 on chromosome 2 and 22 on chromosome 14) by 83 unique peptides following the latest release of neXtProt (2014-09-19). PSMs corresponding to these peptides were thoroughly examined by applying two different MS-based criteria: peptide-level false discovery rate calculation and expert PSM quality assessment. Synthetic peptides were then produced and used to generate reference MS/MS spectra. A spectral similarity score was then calculated for each pair of reference-endogenous spectra and used as a third criterion for missing protein validation. Finally, LC-SRM assays were developed to target proteotypic peptides from four of the missing proteins detected in tissue/cell samples, which were still available and for which sample preparation could be reproduced. These LC-SRM assays unambiguously detected the endogenous unique peptide for three of the proteins. For two of these, identification was confirmed by additional proteotypic peptides. We concluded that of the initial set of 58 proteins detected by the bioinformatics screen, the consecutive MS-based validation criteria led to propose the identification of 13 of these proteins (8 on chromosome 2 and 5 on chromosome 14) that passed at least two of the three MS-based criteria. Thus, a rigorous step-by-step approach combining bioinformatics screening and MS-based validation assays is particularly suitable to obtain protein-level evidence for proteins previously considered as missing. All MS/MS data have been deposited in ProteomeXchange under identifier PXD002131.
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Cromosomas Humanos Par 14 , Cromosomas Humanos Par 2 , Proteínas/genética , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Cromatografía Liquida , Humanos , Datos de Secuencia Molecular , Proteínas/químicaRESUMEN
In bottom-up mass spectrometry-based proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions. Here, we describe the design, synthesis, and validation of a universal protein standard, called DIGESTIF, that can be added to any biological sample. The DIGESTIF standard consists of a soluble recombinant protein scaffold to which a set of 11 artificial peptides (iRT peptides) with good ionization properties has been incorporated. In the protein scaffold, the amino acids flanking iRT peptide cleavage sites were selected either to favor or hinder protease cleavage. After sample processing, the retention time and relative intensity pattern of the released iRT peptides can be used to assess the quality of sample workup, the extent of digestion, and the performance of the LC-MS system. Thus, DIGESTIF can be used to standardize a broad spectrum of applications, ranging from simple replicate measurements to large-scale biomarker screening in biomedical applications.
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Proteínas/química , Proteómica , Secuencia de Aminoácidos , Animales , Biomarcadores/química , Cromatografía Liquida , Humanos , Cinética , Masculino , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Proteolisis , Control de CalidadRESUMEN
A global and rigorous understanding of the signaling pathways and cross-regulatory processes involved in mast cell activation requires the integration of published information with novel functional datasets into a comprehensive computational model. Based on an exhaustive curation of the existing literature and using the software CellDesigner, we have built and annotated a comprehensive molecular map for the FcεRI signaling network. This map can be used to visualize and interpret high-throughput expression data. Furthermore, leaning on this map and using the logical modeling software GINsim, we have derived a qualitative dynamical model, which recapitulates the most salient features of mast cell activation. The resulting logical model can be used to explore the dynamical properties of the system and its responses to different stimuli, in normal or mutant conditions.
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Simulación por Computador , Mastocitos/fisiología , Transducción de Señal/fisiología , Animales , Humanos , Receptores Fc/fisiología , Programas InformáticosRESUMEN
Lysosomes are membrane-bound endocytic organelles that play a major role in degrading cell macromolecules and recycling their building blocks. A comprehensive knowledge of the lysosome function requires an extensive description of its content, an issue partially addressed by previous proteomic analyses. However, the proteins underlying many lysosomal membrane functions, including numerous membrane transporters, remain unidentified. We performed a comparative, semi-quantitative proteomic analysis of rat liver lysosome-enriched and lysosome-nonenriched membranes and used spectral counts to evaluate the relative abundance of proteins. Among a total of 2,385 identified proteins, 734 proteins were significantly enriched in the lysosomal fraction, including 207 proteins already known or predicted as endo-lysosomal and 94 proteins without any known or predicted subcellular localization. The remaining 433 proteins had been previously assigned to other subcellular compartments but may in fact reside on lysosomes either predominantly or as a secondary location. Many membrane-associated complexes implicated in diverse processes such as degradation, membrane trafficking, lysosome biogenesis, lysosome acidification, signaling, and nutrient sensing were enriched in the lysosomal fraction. They were identified to an unprecedented extent as most, if not all, of their subunits were found and retained by our screen. Numerous transporters were also identified, including 46 novel potentially lysosomal proteins. We expressed 12 candidates in HeLa cells and observed that most of them colocalized with the lysosomal marker LAMP1, thus confirming their lysosomal residency. This list of candidate lysosomal proteins substantially increases our knowledge of the lysosomal membrane and provides a basis for further characterization of lysosomal functions.
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Hepatocitos/metabolismo , Membranas Intracelulares/química , Hígado/metabolismo , Lisosomas/química , Proteínas de Transporte de Membrana/aislamiento & purificación , Proteoma/aislamiento & purificación , Animales , Biomarcadores/metabolismo , Expresión Génica , Células HeLa , Hepatocitos/química , Humanos , Hígado/química , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Microscopía Fluorescente , Anotación de Secuencia Molecular , Proteoma/genética , Proteoma/metabolismo , Ratas , Ratas WistarRESUMEN
We report the first proteomic analysis of the SLP76 interactome in resting and activated primary mouse mast cells. This was made possible by a novel genetic approach used for the first time here. It consists in generating knock-in mice that express signaling molecules bearing a C-terminal tag that has a high affinity for a streptavidin analog. Tagged molecules can be used as molecular baits to affinity-purify the molecular complex in which they are engaged, which can then be studied by mass spectrometry. We examined first SLP76 because, although this cytosolic adapter is critical for both T cell and mast cell activation, its role is well known in T cells but not in mast cells. Tagged SLP76 was expressed in physiological amounts and fully functional in mast cells. We unexpectedly found that SLP76 is exquisitely sensitive to mast cell granular proteases, that Zn(2+)-dependent metalloproteases are especially abundant in mast cells and that they were responsible for SLP76 degradation. Adding a Zn(2+) chelator fully protected SLP76 in mast cell lysates, thereby enabling an efficient affinity-purification of this adapter with its partners. Label-free quantitative mass spectrometry analysis of affinity-purified SLP76 interactomes uncovered both partners already described in T cells and novel partners seen in mast cells only. Noticeably, molecules inducibly recruited in both cell types primarily concur to activation signals, whereas molecules recruited in activated mast cells only are mostly associated with inhibition signals. The transmembrane adapter LAT2, and the serine/threonine kinase with an exchange factor activity Bcr were the most recruited molecules. Biochemical and functional validations established the unexpected finding that Bcr is recruited by SLP76 and positively regulates antigen-induced mast cell activation. Knock-in mice expressing tagged molecules with a normal tissue distribution and expression therefore provide potent novel tools to investigate signalosomes and to uncover novel signaling molecules in mast cells.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Mastocitos/metabolismo , Fosfoproteínas/metabolismo , Receptores de IgE/metabolismo , Animales , Células de la Médula Ósea/citología , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mapas de Interacción de Proteínas , Proteómica , Proteínas Proto-Oncogénicas c-bcr/genética , Proteínas Proto-Oncogénicas c-bcr/metabolismoRESUMEN
Cytotoxic T lymphocytes kill target cells via the polarized secretion of cytotoxic granules at the immune synapse. The lytic granules are initially recruited around the polarized microtubule-organizing center. In a dynein-dependent transport process, the granules move along microtubules toward the microtubule-organizing center in the minus-end direction. Here, we found that a kinesin-1-dependent process is required for terminal transport and secretion of polarized lytic granule to the immune synapse. We show that synaptotagmin-like protein 3 (Slp3) is an effector of Rab27a in cytotoxic T lymphocytes and interacts with kinesin-1 through the tetratricopeptide repeat of the kinesin-1 light chain. Inhibition of the Rab27a/Slp3/kinesin-1 transport complex impairs lytic granule secretion. Our data provide further molecular insights into the key functional and regulatory mechanisms underlying the terminal transport of cytotoxic granules and the latter's secretion at the immune synapse.
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Gránulos Citoplasmáticos/metabolismo , Cinesinas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Sinapsis/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas de Unión al GTP rab/metabolismo , Western Blotting , Células Cultivadas , Gránulos Citoplasmáticos/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Cinesinas/antagonistas & inhibidores , Cinesinas/genética , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas del Tejido Nervioso , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTPRESUMEN
Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies.
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Biomarcadores/sangre , Proteínas Sanguíneas/análisis , Enfermedad Coronaria/sangre , Forma MB de la Creatina-Quinasa/sangre , L-Lactato Deshidrogenasa/sangre , Infarto del Miocardio/sangre , Mioglobina/sangre , Troponina I/sangre , Estudios de Casos y Controles , Cromatografía Liquida , Enfermedad Coronaria/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Humanos , Isoenzimas/sangre , Espectrometría de Masas , Infarto del Miocardio/diagnósticoRESUMEN
Absolute quantification of proteins using isotope dilution mass spectrometry requires the selection of proteotypic peptides. When choosing these peptides, a certain number of rules must be respected. Several of these were established to safeguard against quantification errors resulting from the isotopically labeled standard peptides not behaving in the same way as the peptides to be quantified. Of all absolute quantification methods using isotope dilution, Protein Standard for Absolute Quantification (PSAQ(TM) ) offers the maximal protein sequence coverage. In the present study, we show that the PSAQ method presents a previously unreported advantage for protein quantification as it makes use of Met/Cys-containing peptides and peptides-containing miscleavages in addition to proteotypic peptides. By increasing the total number of peptides that can be considered, robustness of quantification is improved, paving the way for a facilitated quantification of low abundant and/or low-molecular-weight proteins.
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Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Péptidos/análisis , Proteómica/métodos , Secuencias de Aminoácidos , Arginina/química , Cisteína/química , Humanos , Marcaje Isotópico , Lisina/química , Metionina/química , Datos de Secuencia Molecular , Péptidos/sangre , Proteolisis , Técnica de Dilución de Radioisótopos , Estándares de Referencia , Tripsina/químicaRESUMEN
Accurate quantification of pure peptides and proteins is essential for biotechnology, clinical chemistry, proteomics, and systems biology. The reference method to quantify peptides and proteins is amino acid analysis (AAA). This consists of an acidic hydrolysis followed by chromatographic separation and spectrophotometric detection of amino acids. Although widely used, this method displays some limitations, in particular the need for large amounts of starting material. Driven by the need to quantify isotope-dilution standards used for absolute quantitative proteomics, particularly stable isotope-labeled (SIL) peptides and PSAQ proteins, we developed a new AAA assay (AAA-MS). This method requires neither derivatization nor chromatographic separation of amino acids. It is based on rapid microwave-assisted acidic hydrolysis followed by high-resolution mass spectrometry analysis of amino acids. Quantification is performed by comparing MS signals from labeled amino acids (SIL peptide- and PSAQ-derived) with those of unlabeled amino acids originating from co-hydrolyzed NIST standard reference materials. For both SIL peptides and PSAQ standards, AAA-MS quantification results were consistent with classical AAA measurements. Compared to AAA assay, AAA-MS was much faster and was 100-fold more sensitive for peptide and protein quantification. Finally, thanks to the development of a labeled protein standard, we also extended AAA-MS analysis to the quantification of unlabeled proteins.
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Aminoácidos/química , Fragmentos de Péptidos/química , Proteínas/química , Secuencia de Aminoácidos , Aminoácidos/análisis , Calibración , Humanos , Hidrólisis , Espectrometría de Masas/normas , Microondas , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Proteínas/análisis , Estándares de Referencia , VolumetríaRESUMEN
The objective of the international Chromosome-Centric Human Proteome Project (C-HPP) is to map and annotate all proteins encoded by the genes on each human chromosome. The C-HPP consortium was established to organize a collaborative network among the research teams responsible for protein mapping of individual chromosomes and to identify compelling biological and genetic mechanisms influencing colocated genes and their protein products. The C-HPP aims to foster the development of proteome analysis and integration of the findings from related molecular -omics technology platforms through collaborations among universities, industries, and private research groups. The C-HPP consortium leadership has elicited broad input for standard guidelines to manage these international efforts more efficiently by mobilizing existing resources and collaborative networks. The C-HPP guidelines set out the collaborative consensus of the C-HPP teams, introduce topics associated with experimental approaches, data production, quality control, treatment, and transparency of data, governance of the consortium, and collaborative benefits. A companion approach for the Biology and Disease-Driven HPP (B/D-HPP) component of the Human Proteome Project is currently being organized, building upon the Human Proteome Organization's organ-based and biofluid-based initiatives (www.hupo.org/research). The common application of these guidelines in the participating laboratories is expected to facilitate the goal of a comprehensive analysis of the human proteome.
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Cromosomas Humanos , Bases de Datos de Proteínas/normas , Proyecto Genoma Humano , Proteoma/análisis , Proteómica/métodos , Proteómica/normas , Guías como Asunto , Humanos , Espectrometría de Masas , Proyectos de InvestigaciónRESUMEN
Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) have been shown to modulate growth factor signaling and follow complex trafficking pathways in neurons. Similarly, several growth factors, including members of the neurotrophin family, undergo axonal retrograde transport that is required to elicit their full signaling potential in neurons. We sought to determine whether IgCAMs that enter the axonal retrograde transport route co-operate with neurotrophin signaling. We identified activated leukocyte cell adhesion molecule (ALCAM), a protein involved in axon pathfinding and development of the neuromuscular junction, to be associated with an axonal endocytic compartment that contains neurotrophins and their receptors. Although ALCAM enters carriers that are transported bidirectionally in motor neuron axons, it is predominantly co-transported with the neurotrophin receptor p75(NTR) toward the cell body. ALCAM was found to specifically potentiate nerve growth factor (NGF)-induced differentiation and signaling. The extracellular domain of ALCAM is both necessary and sufficient to potentiate NGF-induced neurite outgrowth, and its homodimerization is required for this novel role. Our findings indicate that ALCAM synergizes with NGF to induce neuronal differentiation, raising the possibility that it functions not only as an adhesion molecule but also in the modulation of growth factor signaling in the nervous system.
Asunto(s)
Molécula de Adhesión Celular del Leucocito Activado/fisiología , Factores de Crecimiento Nervioso/fisiología , Transducción de Señal/fisiología , Molécula de Adhesión Celular del Leucocito Activado/genética , Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Animales , Transporte Axonal/fisiología , Axones/metabolismo , Western Blotting , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Endosomas/metabolismo , Técnica del Anticuerpo Fluorescente , Espectrometría de Masas , Conformación Molecular , Neuronas Motoras/metabolismo , Factores de Crecimiento Nervioso/farmacología , Neuritas/fisiología , Células PC12 , Fosforilación , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Transducción de Señal/genéticaRESUMEN
After meiosis, during the final stages of spermatogenesis, the haploid male genome undergoes major structural changes, resulting in a shift from a nucleosome-based genome organization to the sperm-specific, highly compacted nucleoprotamine structure. Recent data support the idea that region-specific programming of the haploid male genome is of high importance for the post-fertilization events and for successful embryo development. Although these events constitute a unique and essential step in reproduction, the mechanisms by which they occur have remained completely obscure and the factors involved have mostly remained uncharacterized. Here, we sought a strategy to significantly increase our understanding of proteins controlling the haploid male genome reprogramming, based on the identification of proteins in two specific pools: those with the potential to bind nucleic acids (basic proteins) and proteins capable of binding basic proteins (acidic proteins). For the identification of acidic proteins, we developed an approach involving a transition-protein (TP)-based chromatography, which has the advantage of retaining not only acidic proteins due to the charge interactions, but also potential TP-interacting factors. A second strategy, based on an in-depth bioinformatic analysis of the identified proteins, was then applied to pinpoint within the lists obtained, male germ cells expressed factors relevant to the post-meiotic genome organization. This approach reveals a functional network of DNA-packaging proteins and their putative chaperones and sheds a new light on the way the critical transitions in genome organizations could take place. This work also points to a new area of research in male infertility and sperm quality assessments.
Asunto(s)
Genoma , Meiosis/fisiología , Proteómica/métodos , Animales , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Empaquetamiento del ADN , Redes Reguladoras de Genes , Células Germinativas/citología , Células Germinativas/fisiología , Haploidia , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Espermatogénesis/fisiologíaRESUMEN
In RNA silencing, small RNAs produced by the RNase-III Dicer guide Argonaute-like proteins as part of RNA-induced silencing complexes (RISC) to regulate gene expression transcriptionally or post-transcriptionally. Here, we have characterized the RNA silencing machinery and exhaustive small RNAome of Toxoplasma gondii, member of the Apicomplexa, a phylum of animal- and human-infecting parasites that cause extensive health and economic damages to human populations worldwide. Remarkably, the small RNA-generating machinery of Toxoplasma is phylogenetically and functionally related to that of plants and fungi, and accounts for an exceptionally diverse array of small RNAs. This array includes conspicuous populations of repeat-associated small interfering RNA (siRNA), which, as in plants, likely generate and maintain heterochromatin at DNA repeats and satellites. Toxoplasma small RNAs also include many microRNAs with clear metazoan-like features whose accumulation is sometimes extremely high and dynamic, an unexpected finding given that Toxoplasma is a unicellular protist. Both plant-like heterochromatic small RNAs and metazoan-like microRNAs bind to a single Argonaute protein, Tg-AGO. Toxoplasma miRNAs co-sediment with polyribosomes, and thus, are likely to act as translational regulators, consistent with the lack of catalytic residues in Tg-AGO. Mass spectrometric analyses of the Tg-AGO protein complex revealed a common set of virtually all known RISC components so far characterized in human and Drosophila, as well as novel proteins involved in RNA metabolism. In agreement with its loading with heterochromatic small RNAs, Tg-AGO also associates substoichiometrically with components of known chromatin-repressing complexes. Thus, a puzzling patchwork of silencing processor and effector proteins from plant, fungal and metazoan origin accounts for the production and action of an unsuspected variety of small RNAs in the single-cell parasite Toxoplasma and possibly in other apicomplexans. This study establishes Toxoplasma as a unique model system for studying the evolution and molecular mechanisms of RNA silencing among eukaryotes.
Asunto(s)
Evolución Molecular , Interferencia de ARN/fisiología , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Toxoplasma/genética , Toxoplasmosis/parasitología , Células Cultivadas , Fibroblastos/citología , Fibroblastos/parasitología , Regulación de la Expresión Génica , Genoma de Protozoos , Humanos , MicroARNs/genética , Filogenia , Proteómica , Toxoplasma/crecimiento & desarrolloRESUMEN
Most clock-controlled genes (CCGs) lack the specific E-box response element necessary for direct circadian regulation. This is the case for the prolactin (Prl) gene, the expression of which oscillates in individual lactotrope pituitary cells. To characterize the processes underlying this oscillation, we used a lactotrope cell line (GH4C1 cells). In these cells, Prl gene expression fluctuated significantly during 24 h (P=0.0418). Circadian Prl transcription depended on an interaction between the pituitary-specific transcription factor, PIT-1, and the helicase-like transcription factor (HLTF), a SWI/SNF chromatin remodeler, shown here to bind the Prl promoter on an E-box that differs from the specific E-box preferentially bound by clock proteins. Circadian Prl transcription was further accompanied by marked daily chromatin transitions. While neither HLTF nor PIT-1 was rhythmically expressed, NONO and SFPQ, identified as HLTF-associated proteins by mass spectrometry, displayed a circadian pattern and bound rhythmically to the Prl promoter. Furthermore, NONO and SFPQ were functionally involved in circadian Prl transcription since overexpression of both proteins greatly reduced Prl promoter activity (P<0.001) and disrupted its circadian pattern. A mechanism involving a rhythm in paraspeckle protein recruitment is proposed to explain how the core oscillator can generate a circadian pattern of CCGs lacking the specific E-box response element.