RESUMO
The identification of cell surface proteins on stem cells or stem cell derivatives is a key strategy for the functional characterization, isolation, and understanding of stem cell population dynamics. Here, using an integrated mass spectrometry- and microarray-based approach, we analyzed the surface proteome and transcriptome of cardiac progenitor cells (CPCs) generated from the stage-specific differentiation of mouse and human pluripotent stem cells. Through bioinformatics analysis, we have identified and characterized FZD4 as a marker for lateral plate mesoderm. Additionally, we utilized FZD4, in conjunction with FLK1 and PDGFRA, to further purify CPCs and increase cardiomyocyte (CM) enrichment in both mouse and human systems. Moreover, we have shown that NORRIN presented to FZD4 further increases CM output via proliferation through the canonical WNT pathway. Taken together, these findings demonstrate a role for FZD4 in mammalian cardiac development.
Assuntos
Antígenos de Diferenciação/metabolismo , Proteínas do Olho/metabolismo , Receptores Frizzled/metabolismo , Mesoderma/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Humanos , Mesoderma/citologia , Camundongos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Via de Sinalização WntRESUMO
Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.
Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Segregação de Cromossomos , Histona Desmetilases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Ligação ProteicaRESUMO
Mass spectrometry-based targeted proteomic assays are experiencing a surge in awareness due to the diverse possibilities arising from the re-application of traditional LC-SRM technology. The FDA-approved quantitative LC-SRM-pipeline in drug discovery motivates the use to quantitatively validate putative proteomic biomarkers. However, complexity of biological specimens bears a huge challenge to identify, in parallel, specific peptides and proteins of interest from large biomarker candidate lists. Methods have been devised to increase scan speeds, improve detection specificity and verify quantitative SRM-features. In contrast, high-resolution mass spectrometers could be used to improve reliability and precision of targeted proteomics assays. Here, we present a new method for identifying, quantifying and reporting peptides in high-resolution targeted proteomics experiments performed on an orbitrap hybrid instrument using stable isotope-labeled internal reference peptides. This high precision targeted peptide monitoring (TPM) method has unique advantages over existing techniques, including the need to only detect the most abundant product ion of a given target for confident peptide identification using a scoring function that evaluates assay performance based on 1) m/z-mass accuracy, 2) retention time accuracy of observed species relative to prediction, and 3) retention time accuracy relative to internal reference peptides. Further, we show management of multiplexed precision TPM-assays using sentinel peptide standards. This article is part of a Special Issue entitled: From protein structures to clinical applications.
Assuntos
Espectrometria de Massas , Proteínas Musculares , Miocárdio , Peptídeos , Proteômica , Animais , Biomarcadores/química , Biomarcadores/metabolismo , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Camundongos , Proteínas Musculares/análise , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Miocárdio/química , Miocárdio/metabolismo , Peptídeos/análise , Peptídeos/química , Peptídeos/metabolismo , Proteômica/instrumentação , Proteômica/métodosRESUMO
Single-cell analysis using chemical methods, otherwise known as chemical cytometry, promises to provide significant leaps in understanding signaling processes which result in cellular behavior. Sensitive methods for chemical cytometry such as capillary electrophoresis can detect and quantify multiple targets; however, conclusive identification of detected analytes is required for useful data to be obtained. Here, we demonstrate a method for determining the identity of enzyme-converted peptide products from single cells using a combination of capillary electrophoresis and liquid chromatography-mass spectrometry (LC-MS).
Assuntos
Eletroforese Capilar/métodos , Corantes Fluorescentes/química , Espectrometria de Massas/métodos , Peptídeos/química , Análise de Célula Única/métodos , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia Líquida/métodos , Eletroforese Capilar/instrumentação , Desenho de Equipamento , Humanos , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Análise de Célula Única/instrumentaçãoRESUMO
Bioactive molecules typically mediate their biological effects through direct physical association with one or more cellular proteins. The detection of drug-target interactions is therefore essential for the characterization of compound mechanism of action and off-target effects, but generic label-free approaches for detecting binding events in biological mixtures have remained elusive. Here, we report a method termed target identification by chromatographic co-elution (TICC) for routinely monitoring the interaction of drugs with cellular proteins under nearly physiological conditions in vitro based on simple liquid chromatographic separations of cell-free lysates. Correlative proteomic analysis of drug-bound protein fractions by shotgun sequencing is then performed to identify candidate target(s). The method is highly reproducible, does not require immobilization or derivatization of drug or protein, and is applicable to diverse natural products and synthetic compounds. The capability of TICC to detect known drug-protein target physical interactions (K(d) range: micromolar to nanomolar) is demonstrated both qualitatively and quantitatively. We subsequently used TICC to uncover the sterol biosynthetic enzyme Erg6p as a novel putative anti-fungal target. Furthermore, TICC identified Asc1 and Dak1, a core 40 S ribosomal protein that represses gene expression, and dihydroxyacetone kinase involved in stress adaptation, respectively, as novel yeast targets of a dopamine receptor agonist.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antifúngicos/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Metiltransferases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Sistema Livre de Células , Cromatografia Líquida , Agonistas de Dopamina/metabolismo , Escherichia coli , Proteínas Fúngicas/análise , Proteínas de Ligação ao GTP/análise , Células HeLa , Humanos , Ácidos Hidroxâmicos/metabolismo , Indenos/metabolismo , Macrolídeos/metabolismo , Espectrometria de Massas , Metotrexato/metabolismo , Metiltransferases/análise , Terapia de Alvo Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/análise , Ligação Proteica , Proteômica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/análiseRESUMO
Effective methods to detect and quantify functionally linked regulatory proteins in complex biological samples are essential for investigating mammalian signaling pathways. Traditional immunoassays depend on proprietary reagents that are difficult to generate and multiplex, whereas global proteomic profiling can be tedious and can miss low abundance proteins. Here, we report a target-driven liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy for selectively examining the levels of multiple low abundance components of signaling pathways which are refractory to standard shotgun screening procedures and hence appear limited in current MS/MS repositories. Our stepwise approach consists of: (i) synthesizing microscale peptide arrays, including heavy isotope-labeled internal standards, for use as high quality references to (ii) build empirically validated high density LC-MS/MS detection assays with a retention time scheduling system that can be used to (iii) identify and quantify endogenous low abundance protein targets in complex biological mixtures with high accuracy by correlation to a spectral database using new software tools. The method offers a flexible, rapid, and cost-effective means for routine proteomic exploration of biological systems including "label-free" quantification, while minimizing spurious interferences. As proof-of-concept, we have examined the abundance of transcription factors and protein kinases mediating pluripotency and self-renewal in embryonic stem cell populations.
Assuntos
Cromatografia Líquida/métodos , Peptídeos/química , Análise Serial de Proteínas/métodos , Proteínas/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Camundongos , Peptídeos/síntese química , Peptídeos/genética , Proteômica/métodosRESUMO
Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N- and C-terminal tags and multiple promoters), is compatible with Gateway cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Lentivirus/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteômica/métodos , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Reprogramação Celular/genética , Cromatografia de Afinidade , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Células-Tronco Pluripotentes/citologia , Ligação Proteica , Transcrição GênicaRESUMO
The complex nature of enzyme regulation mandates that enzyme activity profiles be measured in the context of the intact cell. Single-cell capillary electrophoresis (CE) coupled with laser-induced fluorescence is a powerful approach for quantitation and separation of analytes present in small samples and single live cells; however, it does not allow for the definitive identification of the reaction products. On the other hand, mass spectrometry (MS) is able to identify analytes but still lacks the requisite sensitivity for most single-cell analysis applications. Thus, it follows that by determining the relative amounts of reaction products generated in single cells using CE and by producing larger quantities of these products using bulk cell populations to identify them using MS, it is possible to determine enzyme activity profiles in single cells. In this study, the applicability of this approach was demonstrated by examining the intracellular fate of a protease substrate derived from the beta-amyloid precursor protein (beta-APP). In single live TF-1 cells, three distinct fragments were generated from the beta-APP peptide, which differed by a single uncharged amino acid. The CE measurements indicated that the proteolytic fragment profiles (i.e., the relative amounts of each fragment) were consistent from cell to cell but that they were different from those obtained in cell lysates. Furthermore, measurements obtained at the single cell level made it possible to observe a modest but statistically significant negative correlation between the total amount of beta-APP peptide loaded in cells and the fraction of peptide that remained intact. This study demonstrates how single-cell CE, MS, and peptide substrates can be combined to identify and measure enzyme activities in single live cells.
Assuntos
Aminoácidos/análise , Células/metabolismo , Fragmentos de Peptídeos/análise , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/metabolismo , Extratos Celulares , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatografia Líquida , Eletroforese Capilar , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificaçãoRESUMO
The most common cause of kidney transplant failure is the poorly characterized histopathologic entity interstitial fibrosis and tubular atrophy (IFTA). There are no known unifying mechanisms, no effective therapy, and no proven preventive strategies. Possible mechanisms include chronic immune rejection, inflammation, drug toxicity, and chronic kidney injury from secondary factors. To gain further mechanistic insight, we conducted a large-scale proteogenomic study of kidney transplant biopsies with IFTA of varying severity. We acquired proteomic data using tandem mass spectrometry with subsequent quantification, analysis of differential protein expression, validation, and functional annotations to known molecular networks. We performed genome-wide expression profiling in parallel. More than 1400 proteins with unique expression profiles traced the progression from normal transplant biopsies to biopsies with mild to moderate and severe disease. Multiple sets of proteins were mapped to different functional pathways, many increasing with histologic severity, including immune responses, inflammatory cell activation, and apoptosis consistent with the chronic rejection hypothesis. Two examples include the extensive population of the alternative rather than the classical complement pathway, previously not appreciated for IFTA, and a comprehensive control network for the actin cytoskeleton and cell signaling of the acute-phase response. In summary, this proteomic effort using kidney tissue contributes mechanistic insight into several biologic processes associated with IFTA.
Assuntos
Rejeição de Enxerto/genética , Transplante de Rim , Rim/patologia , Proteômica , Adulto , Idoso , Atrofia , Biópsia , Citoesqueleto/fisiologia , Feminino , Fibrose , Rejeição de Enxerto/fisiopatologia , Humanos , Rim/fisiopatologia , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/fisiologiaRESUMO
Recent advances in mass spectrometry and bioinformatics have provided the means to characterize complex protein landscapes from a wide variety of organisms and cell types. Development of standard proteomes exhibiting all of the proteins involved in normal physiology will facilitate the delineation of disease mechanisms. Here, we examine the wild-type cardiac proteome using data obtained from a subcellular fractionation protocol in combination with a multidimensional protein identification proteomics approach. We identified 4906 proteins which were allocated to either cytosolic, microsomal, mitochondrial matrix or mitochondrial membrane fractions with relative abundance values in each fraction. We subjected these proteins to hierarchical clustering, gene ontology terms analysis, immunoblotting, comparison to publicly available protein databases, comparison to 4 distinct cardiac transcriptomes, and finally, to 6 other related proteomic data sets. This study provides an exhaustive analysis of the cardiac proteome and is the first large-scale investigation of the subcellular location for over 2000 unannotated proteins. With the use of a subtractive transcriptomics approach, we have also extended our analysis to identify 'cardiac selective' factors in our proteome. Finally, using specific filtering criteria, we identified proteotypic peptides for subsequent use in targeted studies of both mouse and human. Therefore, we offer this as a major contribution to the advancement of the field of proteomics in cardiovascular research.
Assuntos
Biologia Computacional , Miocárdio/metabolismo , Proteoma/metabolismo , Animais , Feminino , Masculino , CamundongosRESUMO
BACKGROUND: Despite significant improvements in life expectancy of kidney transplant patients due to advances in surgery and immunosuppression, Chronic Allograft Nephropathy (CAN) remains a daunting problem. A complex network of cellular mechanisms in both graft and peripheral immune compartments complicates the non-invasive diagnosis of CAN, which still requires biopsy histology. This is compounded by non-immunological factors contributing to graft injury. There is a pressing need to identify and validate minimally invasive biomarkers for CAN to serve as early predictors of graft loss and as metrics for managing long-term immunosuppression. METHODS: We used DNA microarrays, tandem mass spectroscopy proteomics and bioinformatics to identify genomic and proteomic markers of mild and moderate/severe CAN in peripheral blood of two distinct cohorts (n = 77 total) of kidney transplant patients with biopsy-documented histology. FINDINGS: Gene expression profiles reveal over 2400 genes for mild CAN, and over 700 for moderate/severe CAN. A consensus analysis reveals 393 (mild) and 63 (moderate/severe) final candidates as CAN markers with predictive accuracy of 80% (mild) and 92% (moderate/severe). Proteomic profiles show over 500 candidates each, for both stages of CAN including 302 proteins unique to mild and 509 unique to moderate/severe CAN. CONCLUSIONS: This study identifies several unique signatures of transcript and protein biomarkers with high predictive accuracies for mild and moderate/severe CAN, the most common cause of late allograft failure. These biomarkers are the necessary first step to a proteogenomic classification of CAN based on peripheral blood profiling and will be the targets of a prospective clinical validation study.
Assuntos
Biomarcadores/sangue , Perfilação da Expressão Gênica , Genômica , Nefropatias/sangue , Transplante de Rim , Proteômica , Transplante Homólogo , Cromatografia Líquida , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Espectrometria de Massas em TandemRESUMO
In breast cancer, there is a significant degree of molecular diversity among tumors. Multiple perturbations in signal transduction pathways impinge on transcriptional networks that in turn dictate malignant transformation and metastatic progression. Detailed knowledge of the sequence-specific transcription factors that become activated or repressed within a tumor and comparison of their relative levels of expression in cancer versus normal tissue should therefore provide insight into disease mechanisms, improving patient stratification and facilitating personalized treatment. While high-throughput tandem mass spectrometry methods for global proteome profiling have been developed, existing approaches have limited sensitivity and are often unable to detect low-abundance transcription factors in a complex biological specimen like a biopsy or tumor cell extract. To this end, we have undertaken a systematic comparative evaluation of three MS/MS methods for the ability to detect reference transcription factors spiked in known amounts into a cell-free breast cancer nuclear extract: Data-Dependent Acquisition (DDA), wherein precursor ion intensity dictates selection for fragmentation; Targeted Peptide Monitoring (TPM), a directed approach using successive isolation and fragmentation of predefined m/ z ratios; and Multiple Reaction Monitoring (MRM), in which specific precursor ion to product ion transitions are selectively monitored. Through a series of controlled, parallel benchmarking experiments, we have determined the relative figures-of-merit of each approach, and have established that prior knowledge of signature proteotypic peptides markedly improves overall detection sensitivity, reliability, and quantification.
Assuntos
Neoplasias da Mama/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Fatores de Transcrição/análise , Sequência de Aminoácidos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatografia Líquida , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Subunidade p52 de NF-kappa B/análise , Subunidade p52 de NF-kappa B/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Fator de Transcrição STAT1/análise , Fator de Transcrição STAT1/genética , Fatores de Transcrição/genéticaRESUMO
Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.
Assuntos
Glândula Parótida/química , Proteoma/análise , Saliva/química , Proteínas e Peptídeos Salivares/análise , Glândula Sublingual/química , Glândula Submandibular/química , Adulto , Proteínas Sanguíneas/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise Serial de Proteínas , Lágrimas/químicaRESUMO
Diverse proteomic techniques based on protein MS have been introduced to systematically characterize protein perturbations associated with disease. Progress in clinical proteomics is essential for personalized medicine, wherein treatments will be tailored to individual needs based on patient stratification using noninvasive disease monitoring procedures to reveal the most appropriate therapeutic targets. However, breakthroughs await the successful development and application of a robust proteomic pipeline capable of identifying and rigorously assessing the relevance of multiple candidate proteins as informative diagnostic and prognostic indicators or suitable drug targets involved in a pathological process. While steady progress has been made toward more comprehensive proteome profiling, the emphasis must now shift from in depth screening of reference samples to stringent quantitative validation of selected lead candidates in a broader clinical context. Here, we present an overview of the emerging proteomic strategies for high-throughput protein detection focused primarily on targeted MS/MS as the basis for biomarker verification in large clinical cohorts. We discuss the conceptual promise and practical pitfalls of these methods in terms of achieving higher dynamic range, higher throughput, and more reliable quantification, highlighting research avenues that merit additional inquiry.
RESUMO
Brain metastases are among the most feared complications in breast cancer, as no therapy exists that prevents or eliminates breast cancer spreading to the brain. New therapeutic strategies depend on specific knowledge of tumor cell properties that allow breast cancer cell growth within the brain tissue. To provide information in this direction, we established a human breast cancer cell model for brain metastasis based on circulating tumor cells from a breast cancer patient and variants of these cells derived from bone or brain lesions in immunodeficient mice. The brain-derived cells showed an increased potential for brain metastasis in vivo and exhibited a unique protein expression profile identified by large-scale proteomic analysis. This protein profile is consistent with either a selection of predisposed cells or bioenergetic adaptation of the tumor cells to the unique energy metabolism of the brain. Increased expression of enzymes involved in glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation pathways suggests that the brain metastatic cells derive energy from glucose oxidation. The cells further showed enhanced activation of the pentose phosphate pathway and the glutathione system, which can minimize production of reactive oxygen species resulting from an enhanced oxidative metabolism. These changes promoted resistance of brain metastatic cells to drugs that affect the cellular redox balance. Importantly, the metabolic alterations are associated with strongly enhanced tumor cell survival and proliferation in the brain microenvironment. Thus, our data support the hypothesis that predisposition or adaptation of the tumor cell energy metabolism is a key element in breast cancer brain metastasis, and raise the possibility of targeting the functional differentiation in breast cancer brain lesions as a novel therapeutic strategy.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Animais , Processos de Crescimento Celular/fisiologia , Ciclo do Ácido Cítrico , Modelos Animais de Doenças , Metabolismo Energético , Feminino , Glutationa/metabolismo , Glicólise , Humanos , Camundongos , Camundongos SCID , Mitocôndrias/metabolismo , Oxirredução , Consumo de Oxigênio , Via de Pentose Fosfato , ProteômicaRESUMO
Specific identification of Bacillus anthracis (B. anthracis) is vital for the accurate treatment of afflicted personnel during biological warfare situations and civilian terrorist attacks. In order to accomplish this, we have subjected the lysates from B. anthracis to affinity purification using monoclonal antibodies for the selected antigenic protein present in the bacteria. The bound antigenic protein was identified by multi-dimensional protein identification technology (MudPIT) to be a surface layer protein EA1. The same antigen was identified from the lysates from a few strains of B. anthracis demonstrating the observation to be common for B. anthracis strains. Hence, this presents an effective pathway for the identification of the bacteria present in unknown samples of various origins. Generation of a database containing the EA1 protein has been found to be useful in the database search of unknown samples.
Assuntos
Bacillus anthracis/química , Marcadores de Afinidade , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos , Anticorpos Monoclonais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Bacillus anthracis/classificação , Bacillus anthracis/genética , Bacillus anthracis/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Guerra Biológica , Bioterrorismo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Espectrometria de Massas em Tandem/métodosRESUMO
Endothelial progenitor cells (EPCs) have significant therapeutic potential. However, the low quantity of such cells available from bone marrow and their limited capacity to proliferate in culture make their use difficult. Here, we present the first definitive demonstration of the presence of true EPCs in murine fetal liver capable of forming blood vessels in vivo connected to the host's vasculature after transplantation. This population is particularly interesting because it can be obtained at high yield and has a high angiogenic capacity as compared with bone marrow-derived EPCs. The EPC capacity is contained within the CD31+Sca1+ cell subset. We demonstrate that these cells are dependent for survival and proliferation on a feeder cell monolayer derived from the fetal liver. In addition, we describe a novel and easy method for the isolation and ex vivo proliferation of these EPCs. Finally, we used gene expression profiling and tandem mass spectrometry proteomics to examine the fetal liver endothelial progenitors and the feeder cells to identify possible proangiogenic growth factor and endothelial differentiation-associated genes.
Assuntos
Células Endoteliais/fisiologia , Fígado/citologia , Neovascularização Fisiológica , Células-Tronco/fisiologia , Animais , Antígenos Ly/genética , Diferenciação Celular , Separação Celular , Perfilação da Expressão Gênica , Técnicas In Vitro , Fígado/irrigação sanguínea , Fígado/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Neovascularização Fisiológica/genética , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Transplante de Células-TroncoRESUMO
In the past decade, shotgun proteomic analysis has been utilized extensively to answer complex biological questions. New challenges arise in large scale proteomic profiling when dealing with complex biological mixtures such as the mammalian cell lysate. In this study, we explored the approach of protein separation prior to the shotgun multidimensional protein identification technology (MudPIT) analysis. We fractionated the mammalian cancer cell lysate using the PF 2D ProteomeLab system and analyzed the distribution of molecular weight, isoelectric point, and cellular localization of the eluted proteins. As a result, we were able to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins with lower abundance in the complex protein mixture.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/química , Proteínas de Neoplasias/análise , Proteômica/métodos , Análise de Sequência de Proteína , Biomarcadores Tumorais/isolamento & purificação , Neoplasias da Mama/secundário , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Eletroforese em Gel Bidimensional , Feminino , Humanos , Focalização Isoelétrica , Proteínas de Neoplasias/isolamento & purificação , Proteômica/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais CultivadasRESUMO
Four phosphoenolpyruvate (PEP) derivatives, carrying reactive or activable chemical functions in each of the three chemical regions of PEP, were assayed as alternative substrates of enzyme I (EI) of the Escherichia coli PEP:glucose phosphotransferase system. The Z- and E-isomers of 3-chlorophosphoenolpyruvate (3-Cl-PEP) were substrates, presenting K(m) values of 0.08 and 0.12 mm, respectively, very similar to the K(m) of 0.14 mm measured for PEP, and k(cat) of 40 and 4 min(-1), compared with 2,200 min(-1), for PEP. The low catalytic efficiency of these substrates permits the study of activity at in vivo EI concentrations. Z-Cl-PEP was a competitive inhibitor of PEP with a K(I) of 0.4 mm. E-Cl-PEP was not an inhibitor. Compounds 3 and 4, obtained by modification of the carboxylic and phosphate groups of PEP, were neither substrates nor inhibitors of EI, highlighting the importance of these functionalities for recognition by EI. Z-Cl-PEP is a suicide inhibitor. About 10-50 turnovers sufficed to inactivate EI completely. Such a property can be exploited to reveal and quantitate phosphoryl transfer from EI to other proteins at in vivo concentrations. Inactivation was saturatable in Z-Cl-PEP, with an apparent K(m)(inact) of 0.2-0.4 mm. The rate of inactivation increased with the concentration of EI, indicating a preferential or exclusive reaction with the dimeric form of EI. E-Cl-PEP inactivates EI much more slowly, and unlike PEP, it did not protect against inactivation by Z-Cl-PEP. This and the ineffectiveness of E-Cl-PEP as a competitive inhibitor have been related to the presence of two EI active species. Cys-502 of EI was identified by mass spectrometry as the reacting residue. The C502A EI mutant showed less than 0.06% wild-type activity. Sequence alignments and comparisons of x-ray structures of different PEP-utilizing enzymes indicate that Cys-502 might serve as a proton donor during catalysis.