RESUMEN
Hepatocellular carcinoma (HCC) has been ranked as the third leading cause of cancer-related mortality worldwide. Typically, patients are already in advanced stages of liver cirrhosis at the time of HCC diagnosis. Because HCC is often detected at a late stage and is highly aggressive, noninvasive biomarkers are urgently needed for early diagnosis. Recent advances in gene-expression profiling technologies have enabled molecular classification of HCC into defined subclasses that provide a firm basis for further study of potential mechanisms and biomarkers underlying the development of HCC. This study applied an integrated onco-proteogenomics approach to identify and characterize HCC biomarkers. Specifically, this study integrated proteomic, genomic, and transcriptomic methods to obtain protein expression profiles of urine and tissue samples from HCC patients and from normal controls. Two mediators of inflammation were positively identified: S100A9 and granulin protein markers, which belong to the cytoplasmic alarmin family of the host innate immune system. These HCC-associated cancer-specific biomarkers may have contributing roles not only in the dysregulated processes associated with various inflammatory and autoimmune conditions, but also in tumorigenesis and cancer metastasis.
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Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Proteogenómica/métodos , Investigación Biomédica Traslacional , Humanos , Espectrometría de MasasRESUMEN
OBJECTIVES: The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. METHODS: Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. RESULTS: Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥ 2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 ß, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. CONCLUSIONS: The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC.
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Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteómica , Animales , Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Cromatografía Liquida , Biología Computacional , Bases de Datos de Proteínas , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Marcaje Isotópico , Ratones , Ratones Transgénicos , Nanotecnología , Proteínas de Neoplasias/genética , Estadificación de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Mapeo Peptídico , Fenotipo , Mapas de Interacción de Proteínas , Proteómica/métodos , Espectrometría de Masas en Tándem , Factores de TiempoRESUMEN
Hepatocellular carcinoma (HCC), the major type of liver cancer, is among the most lethal cancers owing to its aggressive nature and frequently late detection. Therefore, the possibility to identify early diagnostic markers could be of significant benefit. Urine has especially become one of the most attractive body fluids in biomarker discovery as it can be obtained non-invasively in large quantities and is stable as compared with other body fluids. To identify potential protein biomarker for early diagnosis of HCC, we explored protein expression profiles in urine from HCC patients and normal controls (n = 44) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry (nanoLC-MS/MS) and stable isotope dimethyl labeling. We have systematically mapped 91 proteins with differential expressions (p < 0.05), which included 8 down-regulated microtubule proteins and 83 up-regulated proteins involved in signal and inflammation response. Further integrated proteogenomic approach composed of proteomic, genomic and transcriptomic analysis identified that S100A9 and GRN were co-amplified (p < 0.001) and co-expressed (p < 0.01) in HCC tumors and urine samples. In addition, the amplifications of S100A9 or GRN were found to be associated with poor survival in HCC patients, and their co-amplification was also prognosed worse overall survival than individual ones. Our results suggest that urinary S100A9 and GRN as potential combinatorial biomarkers can be applied to early diagnosis of hepatocellular carcinoma and highlight the utility of onco-proteogenomics for identifying protein markers that can be applied to disease-oriented translational medicine.
RESUMEN
The beneficial effects of garlic (Allium sativum) consumption in treating human diseases have been reported worldwide over a long period of human history. The strong antioxidant effect of garlic extract (GE) has also recently been claimed to prevent cancer, thrombus formation, cardiovascular disease and some age-related maladies. Using Caenorhabditis elegans as a model organism, aqueous GE was herein shown to increase the expression of longevity-related FOXO transcription factor daf-16 and extend lifespan by 20%. By employing microarray and proteomics analysis on C. elegans treated with aqueous GE, we have systematically mapped 229 genes and 46 proteins with differential expression profiles, which included many metabolic enzymes and yolky egg vitellogenins. To investigate the garlic components functionally involved in longevity, an integrated metabolo-proteomics approach was employed to identify metabolites and protein components associated with treatment of aqueous GE. Among potential lifespan-promoting substances, mannose-binding lectin and N-acetylcysteine were found to increase daf-16 expression. Our study points to the fact that the lifespan-promoting effect of aqueous GE may entail the DAF-16-mediated signaling pathway. The result also highlights the utility of metabolo-proteomics for unraveling the complexity and intricacy involved in the metabolism of natural products in vivo.
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Ajo/química , Longevidad/efectos de los fármacos , Metabolómica/métodos , Extractos Vegetales/farmacología , Proteómica/métodos , Acetilcisteína/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografía Liquida , Clonación Molecular , Regulación hacia Abajo , Evolución Molecular , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Lectina de Unión a Manosa/metabolismo , Datos de Secuencia Molecular , Transducción de Señal , Espectrometría de Masas en Tándem , Regulación hacia Arriba , Vitelogeninas/genética , Vitelogeninas/metabolismoRESUMEN
Inflammation plays a key role in coronary artery disease (CAD) and other manifestations of atherosclerosis. Recently, urinary proteins were found to be useful markers for reflecting inflammation status of different organs. To identify potential biomarker for diagnosis of CAD, we performed one-dimensional SDS-gel electrophoresis followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Among the proteins differentially expressed in urine samples, monocyte antigen CD14 was found to be consistently expressed in higher amounts in the CAD patients as compared to normal controls. Using enzyme-linked immunosorbent assays to analyze the concentrations of CD14 in urine and serum, we confirmed that urinary CD14 levels were significantly higher in patients (n = 73) with multi-vessel and single vessel CAD than in normal control (n = 35) (P < 0.001). Logistic regression analysis further showed that urinary CD14 concentration level is associated with severity or number of diseased vessels and SYNTAX score after adjustment for potential confounders. Concomitantly, the proportion of CD14+ monocytes was significantly increased in CAD patients (59.7 ± 3.6%) as compared with healthy controls (14.9 ± 2.1%) (P < 0.001), implicating that a high level of urinary CD14 may be potentially involved in mechanism(s) leading to CAD pathogenesis. By performing shotgun proteomics, we further revealed that CD14-associated inflammatory response networks may play an essential role in CAD. In conclusion, the current study has demonstrated that release of CD14 in urine coupled with more CD14+ monocytes in CAD patients is significantly correlated with severity of CAD, pointing to the potential application of urinary CD14 as a novel noninvasive biomarker for large-scale diagnostic screening of susceptible CAD patients.
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Biomarcadores/orina , Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/orina , Receptores de Lipopolisacáridos/orina , Proteoma/metabolismo , Anciano , Estudios de Casos y Controles , Enfermedad de la Arteria Coronaria/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/orina , Monocitos/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
The development of gastrointestinal diseases has been found to be associated with Helicobacter pylori (H. pylori) infection and various biochemical stresses in stomach and intestine. These stresses, such as oxidative, osmotic and acid stresses, may bring about bi-directional effects on both hosts and H. pylori, leading to changes of protein expression in their proteomes. Therefore, proteins differentially expressed in H. pylori under various stresses not only reflect gastrointestinal environment but also provide useful biomarkers for disease diagnosis and prognosis. In this regard, proteomic technology is an ideal tool to identify potential biomarkers as it can systematically monitor proteins and protein variation on a large scale of cell's translational landscape, permitting in-depth analyses of host and pathogen interactions. By performing two-dimensional polyacrylamide gel electrophoresis (2-DE) followed by liquid chromatography-nanoESI-mass spectrometry (nanoLC-MS/MS), we have successfully pinpointed alkylhydroperoxide reductase (AhpC), neutrophil-activating protein and non-heme iron-binding ferritin as three prospective biomarkers showing up-regulation in H. pylori under oxidative, osmotic and acid stresses, respectively. Further biochemical characterization reveals that various environmental stresses can induce protein structure change and functional conversion in the identified biomarkers. Especially salient is the antioxidant enzyme AhpC, an abundant antioxidant protein present in H. pylori. It switches from a peroxide reductase of low-molecular-weight (LMW) oligomers to a molecular chaperone of high-molecular-weight (HMW) complexes under oxidative stress. Different seropositivy responses against LMW or HMW AhpC in H. pylori-infected patients faithfully match the disease progression from disease-free healthy persons to patients with gastric ulcer and cancer. These results has established AhpC of H. pylori as a promising diagnostic marker for gastrointestinal maladies, and highlight the utility of clinical proteomics for identifying disease biomarkers that can be uniquely applied to disease-oriented translational medicine.
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Biomarcadores/metabolismo , Enfermedades Gastrointestinales/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/metabolismo , Proteómica/métodos , Ácidos/química , Proteínas Bacterianas/metabolismo , Electroforesis en Gel Bidimensional , Ambiente , Ferritinas/metabolismo , Infecciones por Helicobacter/diagnóstico , Humanos , Inflamación , Ósmosis , Estrés Oxidativo , Peroxirredoxinas/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Although morphological and anatomical studies indicate that varicose veins are characterized by venous wall weakening and subendothelial fibrosis, the exact underlying biochemical mechanism of their development remains unknown. Additionally, no quantitative proteomic study of venous proteins leading to decreased contractility of varicose veins has been reported to date. Therefore, to elucidate the molecular mechanism of altered vascular contractility, this study performed shotgun proteomic analysis to obtain protein expression profiles in patients with varicose veins. Stable isotope dimethyl labeling coupled with nanoLC-MS/MS revealed downregulation in 12 polypeptides, including myosin light chain kinase, creatine kinase B-type, ATP synthase, phosphoglycerate kinase, and pyruvate kinase. However, analyses of protein species associated with cytoskeletal assembly or with cellular morphology showed no clear up- or down-regulation. These results indicate that defects in ATP generation and utilization may account for the dysfunction of vascular smooth muscle following formation of varicose veins. Collectively, the severity of varicose veins depends on the regulatory roles of various protein factors in the metabolic coordination of physiological functions. This pilot study improves understanding of the pathogenesis of varicose veins and lays the foundation for further validation and clinical translation of biomarkers for targeted therapies in treating this disease.
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Adenosina Trifosfato/biosíntesis , Proteínas/metabolismo , Proteómica , Várices/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Cromatografía Liquida , Humanos , Datos de Secuencia Molecular , Proteínas/química , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
PURPOSE: The aim of this study was to determine the lens crystallin diversity of degenerative eyes from the rice eel (Monopterus albus) and walking catfish (Clarias batrachus) as compared to that of zebrafish (Danio rerio) by using comparative proteomics methodologies. We endeavored to investigate the evolution of vertebrate lenses particularly concerning the functional loss of lenses in degenerative eyes of rice eels and catfishes living under an environment of perpetual darkness. METHODS: Fish lenses were collected and homogenized to extract total soluble proteins. The protein mixtures were separated by one- and two-dimensional gel electrophoresis (1D or 2D gel), plus the newer gel-free shotgun proteomic strategy, followed by in-gel digestion and subjection of the digested protein bands or spots to liquid chromatography coupled with tandem mass spectrometry. The proteomics data were analyzed and compared based on the proteomics databank of zebrafish. The soluble lens protein solutions of three piscine species were also processed by gel-filtration chromatography and 1D sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the comparison and validation of various crystallin families, e.g., α-, ß-, and γ-crystallins. RESULTS: In zebrafish eye lenses, γ-crystallin constituted about 71% and α- and ß-crystallins comprised 30% of total lens proteins. In rice eel lenses, very little or almost no α-crystallins were detected and ß- and γ-crystallins comprised more than 98% of total lens proteins. In catfish lenses, α- and ß-crystallins comprised about 40% and γ-crystallin constitutes 60% of total lens proteins. It was of interest to find that α-crystallin was totally absent in the rice eel in contrast to the presence, albeit with very low amounts, of α-crystallin in similarly nocturnal catfish. The ratio of α-crystallin subunits (αA/αB) was found to be about 20:1 for the catfish lens, in great contrast to the ratio of about 3:1 found for most mammalian lenses. In contrast, ß- and γ-crystallins were more abundant in lenses of these three piscine species, similar to mammalian lenses. By proteomics analysis, the most abundant ß-crystallins were found to comprise a diverse group of ßA1a, ßA1-2, ßA2a, ßA2-2, ßA4, ßB1, ßB2, and ßB3 subunit crystallins; the monomeric γ-crystallin class contains γB, γD, γM2, γM3, γM5, γM7, γN-A, γN-B, γS1, and γS2 crystallins. CONCLUSIONS: In cave or nocturnal animals, the eye is sometimes reduced or eliminated because of adaptation to life in visual darkness. The comparative proteomics analysis of degenerative and normal lenses forms a firm molecular basis to investigate further the evolution of piscine lenses in the future. The total numbers of α-, ß-, and γ-crystallins in the three fish species as revealed by the current proteomics methodology clearly indicate the complexity and diversity of crystallin species present in the piscine class of vertebrates. The unexpected finding that α-crystallin is absent in the degenerative eye lenses of rice eel may have some bearing on the chaperone function of α-crystallin in regard to its protective role of preventing protein aggregation in diurnal vertebrate lenses to maintain functional transparency.
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Bagres/metabolismo , Ritmo Circadiano , Anguilas/metabolismo , Cristalino/metabolismo , Cristalino/patología , Proteómica/métodos , Pez Cebra/metabolismo , Animales , Tamaño Corporal , Bagres/anatomía & histología , Extractos Celulares , Fraccionamiento Químico , Cromatografía en Gel , Cristalinas/metabolismo , Anguilas/anatomía & histología , Electroforesis en Gel Bidimensional , Proteínas del Ojo/metabolismo , Proteínas de Peces/metabolismo , Cristalino/anatomía & histología , Oryza , Pez Cebra/anatomía & histologíaRESUMEN
It is generally accepted that most gastrointestinal diseases are probably caused by the bacterial pathogen Helicobacter pylori (H. pylori). In this study we have focused on the comparison of protein expression profiles of H. pylori grown under normal and high-salt conditions by a proteomics approach. We have identified about 190 proteins whose expression levels changed after growth at high salt concentration. Among these proteins, neutrophil-activating protein (NapA) was found to be consistently up-regulated under osmotic stress brought by high salts. We have investigated the effect of high salt on secondary and tertiary structures of NapA by circular dichroism spectroscopy followed by analytical ultracentrifugation to monitor the change of quaternary structure of recombinant NapA with increasing salt concentration. The loss of iron-binding activity of NapA coupled with noticeable energetic variation in protein association of NapA as revealed by isothermal titration calorimetry was found under high salt condition. The phylogenetic tree analysis based on sequence comparison of 16 protein sequences encompassing NapA proteins and ferritin of H. pylori and other prokaryotic organisms pointed to the fact that all H. pylori NapA proteins of human origin are more homologous to NapA of Helicobacter genus than to other bacterial NapA. Based on computer modeling, NapA proteins from H. pylori of human isolates are found more similar to ferritin from H. pylori than to NapA from other species of bacteria. Taken together, these results suggested that divergent evolution of NapA and ferritin possessing dissimilar and diverse sequences follows a path distinct from that of convergent evolution of NapA and ferritin with similar dual functionality of iron-binding and ferroxidase activities.
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Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Ferritinas/genética , Ferritinas/metabolismo , Helicobacter pylori/metabolismo , Filogenia , Secuencia de Aminoácidos , Evolución Biológica , Electroforesis en Gel Bidimensional , Ferritinas/química , Infecciones por Helicobacter/genética , Helicobacter pylori/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Presión Osmótica/fisiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Regulación hacia ArribaRESUMEN
OBJECTIVES: AlphaB-crystallin (αB-crystallin), a small heat shock protein, has been reported to be involved in the growth, antiapoptosis, migration, and chemoresistance of human malignancies. MATERIALS AND METHODS: αB-crystallin expression in normal renal and clear cell renal cell carcinoma (ccRCC) tissues was examined with two-dimensional (2D) gel electrophoresis assays. Immunohistochemistry was conducted to determine the presence of αB-crystallin-positive tumor cells and staining intensity in 50 cases of ccRCC tissue samples. The association of αB-crystallin protein expression, clinicopathogic parameters and prognosis of ccRCC patients was also analyzed with Student's t-test and Kaplan-Meier analysis. Moreover, Western blot assays were performed to detect the protein expression of αB-crystallin in normal and tumor tissues and the alteration of cell cycle regulators in αB-crystallin-overexpressing cells. MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide), BrdU, and transwell assays were performed to demonstrate the effects of αB-crystallin overexpression on cell growth, DNA synthesis and cell migration of ccRCC cells, respectively. RESULTS: The results showed the up-regulation of αB-crystallin expression in ccRCC tissues. Overall survival of ccRCC patients was significantly correlated with αB-crystallin expression in tumor tissues. We found that αB-crystallin overexpression increased the expression of cyclin A and the incorporation of BrdU, which may be related to the enhancement of cell growth. Transwell analyses demonstrated that presence of αB-crystallin overexpression enhanced cell migration in ccRCC cells. Furthermore, rapamycin-resistance of tumor cells was induced when αB-crystallin was overexpressed. CONCLUSIONS: Our experimental findings highlight the importance of αB-crystallin in the tumor growth, migration, and target therapy-resistance of ccRCC cells.
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Carcinoma de Células Renales/metabolismo , Neoplasias Renales/metabolismo , Cadena B de alfa-Cristalina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Ciclina A/metabolismo , Progresión de la Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Electroforesis en Gel Bidimensional , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Pronóstico , Proteómica/métodos , Sirolimus/farmacologíaRESUMEN
The development of various gastrointestinal diseases was suggested to be associated with chronic inflammation as a consequence of Helicobacter pylori (H. pylori) infection. Our previous studies showed that an antioxidant protein alkylhydroperoxide reductase (AhpC) is an abundant and important antioxidant protein present in H. pylori. In this study we have explored the potential of utilizing antibodies to AhpC for detection of patients who are at high risks of evolving into severe outcomes of gastric malignancies after H. pylori infection. The correlation between AhpC and extents of inflammatory damage in tissues was demonstrated by immunoblotting assays and endoscopic examinations. Oxidative stress-induced high-molecular-weight (HMW) AhpC with chaperone activity in vivo was further investigated by co-immunoprecipitation, 2-dimensional gel electrophoresis (2-DE) followed by nano-liquid chromatography coupled tandem mass spectrometry (nanoLC-MS/MS). We found AhpC was consistently expressed in higher amounts in H. pylori strains isolated from patients with gastric cancer (GC) than gastritis (GA). Immunological analysis of seropositivity for AhpC indicated that positive diagnostic rates for H. pylori-infected patients with GA, gastric ulcer (GU) and GC were 68% (15/22), 100% (50/50) and 100% (50/50), respectively. In great contrast to low-molecular-weight (LMW) AhpC, HMW AhpC with chaperone function was found to distribute inside of H. pylori cells. We also found that LMW forms of AhpC were recognized by serum antibodies from GA patients whereas HMW forms of AhpC reacted mainly with those from GU and GC patients. Based on the significant difference between AhpC isolated from strains of GC and GA, it is conceivable that AhpC of H. pylori may prove to be useful as a prognostic or diagnostic protein marker to monitor varied clinical manifestations of gastrointestinal patients infected with H. pylori.
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Proteínas Bacterianas/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter pylori/enzimología , Peroxirredoxinas/metabolismo , Proteínas Bacterianas/genética , Biomarcadores/metabolismo , Electroforesis en Gel Bidimensional , Gastritis/diagnóstico , Gastritis/microbiología , Genotipo , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori/genética , Helicobacter pylori/ultraestructura , Humanos , Immunoblotting , Microscopía Electrónica de Transmisión , Chaperonas Moleculares/metabolismo , Peso Molecular , Estrés Oxidativo , Peroxirredoxinas/química , Peroxirredoxinas/genética , Úlcera Gástrica/diagnóstico , Úlcera Gástrica/microbiologíaRESUMEN
This account will give an overview and evaluation of the current advances in mass spectrometry (MS)-based proteomics platforms and technology. A general review of some background information concerning the application of these methods in the characterization of molecular sizes and related protein expression profiles associated with different types of cells under varied experimental conditions will be presented. It is intended to provide a concise and succinct overview to those clinical researchers first exposed to this foremost powerful methodology in modern life sciences of postgenomic era. Proteomic characterization using highly sophisticated and expensive instrumentation of MS has been used to characterize biological samples of complex protein mixtures with vastly different protein structure and composition. These systems are then used to highlight the versatility and potential of the MS-based proteomic strategies for facilitating protein expression analysis of various disease-related organisms or tissues of interest. Major MS-based strategies reviewed herein include (1) matrix-assisted laser desorption ionization-MS and electron-spray ionization proteomics; (2) one-dimensional or two-dimensional gel-based proteomics; (3) gel-free shotgun proteomics in conjunction with liquid chromatography/tandem MS; (4) Multiple reaction monitoring coupled tandem MS quantitative proteomics and; (5) Phosphoproteomics based on immobilized metal affinity chromatography and liquid chromatography-MS/MS.
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Proteómica/métodos , Cromatografía Liquida , Humanos , Espectrometría de Masas , Espectrometría de Masas en TándemRESUMEN
PURPOSE: Post-translational modification (PTM) of lens proteins is believed to play various roles in age-related lens function and development. Among the different types of PTM, phosphorylation is most noteworthy to play a major role in the regulation of various biosignaling pathways in relation to metabolic processes and cellular functions. The present study reported the quantitative analysis of the in vivo phosphoproteomics profiles of human normal and cataractous lenses with the aim of identifying specific phosphorylation sites which may provide insights into the physiologic significance of phosphorylation in relation to cataract formation. METHODS: To improve detection sensitivity of low abundant proteins, we first adopted SDS-gel electrophoresis fractionation of lens extracts to identify and compare the protein compositions between normal and cataractous lenses, followed by tryptic digestion, enrichment of phosphopeptides by immobilized metal affinity chromatography (IMAC) and nano-liquid chromatography coupled tandem mass spectrometry (nanoLC-MS/MS) analysis. RESULTS: By comprehensively screening of the phosphoproteome in normal and cataractous lenses, we identified 32 phosphoproteins and 73 phosphorylated sites. The most abundantly phosphorylated proteins are two subunits of ß-crystallin, i.e., ßB1-crystallin (12%) and ßB2-crystallin (12%). Moreover, serine was found to be the most abundantly phosphorylated residue (72%) in comparison with threonine (24%) and tyrosine (4%) in the lens phosphoproteome. The quantitative analysis revealed significant and distinct changes of 19 phosphoproteins corresponding to 28 phosphorylated sites between these two types of human lenses, including 20 newly discovered novel phosphorylation sites on lens proteins. CONCLUSIONS: The shotgun phosphoproteomics approach to characterize protein phosphorylation may be adapted and extended to the comprehensive analysis of other types of post-translational modification of lens proteins in vivo. The identification of these novel phosphorylation sites in lens proteins that showed differential expression in the cataractous lens may bear some unknown physiologic significance and provide insights into phosphorylation-related human eye diseases, which warrant further investigation in the future.
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Catarata/metabolismo , Cristalino/metabolismo , Fosfoproteínas/química , Proteómica/métodos , Adulto , Anciano , Cromatografía Liquida/métodos , Cristalinas/metabolismo , Humanos , Fosforilación , Serina/química , Transducción de Señal , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Tripsina/farmacología , Tirosina/químicaRESUMEN
The development of gastric cancer was suggested to be associated with chronic inflammation as a consequence of Helicobacter pylori infection. Such inflammation-related oxidative stress induced by reactive oxygen species (ROS) in vivo may exert bidirectional effects on both hosts and H pylori. In this study, ROS-induced oxidative stress was mimicked by coculture of gastric epithelial cells with H pylori treated with hydrogen peroxide (H(2)O(2)). To investigate the effect of H(2)O(2) on the proteome of H pylori, we performed two-dimensional polyacrylamide gel electrophoresis followed by liquid chromatography coupled with nano-electrospray ionization-tandem mass spectrometry (liquid chromatography mass spectrometry) and bioinformatics database analysis. The nine most overexpressed proteins consisted of three virulence factors, including cytotoxin-associated protein A (CagA), vacuolating cytotoxin (VacA), adherence-associated protein (AlpA), and two antioxidant enzymes alkylhydroperoxide reductase (AhpC) and catalase (KatA), plus one serine protease (HtrA), aconitate hydratase, and fumarate reductase. We have also confirmed the upregulation of virulence factors and antioxidant proteins in several H pylori strains isolated from patients of different clinical outcomes. Furthermore, it is noted that H pylori was found to decrease in infection rate and increase in proliferation after being exposed to H(2)O(2). We also found that gastric epithelial cells can be protected from oxidative damage by H(2)O(2) in the presence of H pylori. In conclusion, this study lends support to the supposition that ROS containing H(2)O(2) as one of the major oxidative species can induce upregulation of virulence factors and antioxidant enzymes in H pylori, which may aid in the elucidation of inflammation leading to the development of gastric cancer from H pylori infection.
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Proteínas Bacterianas/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter pylori/metabolismo , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Estrés Oxidativo , Regulación hacia Arriba , Proteínas Bacterianas/genética , Línea Celular Tumoral , Técnicas de Cocultivo , Regulación Bacteriana de la Expresión Génica , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/aislamiento & purificación , Humanos , Proteómica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/microbiología , Transcripción Genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
PURPOSE: Phosphorylation is an important post-translational modification for the cellular regulation of various biosignaling pathways. We have identified in vivo phosphorylation sites of various lens proteins including especially the major structural proteins of the crystallin family from porcine eye lenses by means of two-dimensional gel electrophoresis (2-DE) or immobilized metal affinity chromatography (IMAC) followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). METHODS: For the identification of phosphorylated residues in various lens proteins of porcine lens extracts, we have adapted two complementary proteomic approaches, i.e., pre-fractionation of protein samples with 2-DE or enrichment of phosphopeptides with IMAC followed by LC-MS/MS analysis and database search. The results were compared and validated with those in phosphoproteomics databases. RESULTS: Two subunits of alpha-crystallin, alphaA-crystallin and alphaB-crystallin, as well as other lens crystallins and non-crystallin cellular proteins, such as beta-enolase, heat shock protein beta-1 (HSP27), and glucose-6-phosphate isomerase (GPI) were found to be phosphorylated in vivo at specific sites. Moreover, alphaA- and alphaB-crystallins were found to be the most abundantly phosphorylated proteins in porcine lenses, being extensively phosphorylated on serine or threonine, but not on tyrosine residues. CONCLUSIONS: The complementary gel-based and gel-free proteomic strategies have been compared and evaluated for the study of crystallin phosphorylation from whole tissue extracts of porcine eye lenses. Technically, the IMAC method facilitates direct site-specific identification of phosphorylation residues in lens proteins, which does not necessitate the pre-MS/MS 2-DE separation of protein samples. Moreover, the improved strategy using gel-free phosphoproteomics analysis affords a more effective and simplistic method for the determination of in vivo phosphorylation sites than the conventional 2-DE pre-separation of protein mixture. This study should form a firm basis for the comprehensive analysis of post-translational modification of lens proteins in terms of aging or various diseased states.
Asunto(s)
Cristalinas/química , Cristalinas/metabolismo , Cristalino/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Animales , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Glucosa-6-Fosfato Isomerasa/química , Glucosa-6-Fosfato Isomerasa/metabolismo , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/metabolismo , Datos de Secuencia Molecular , Fosfopiruvato Hidratasa/química , Fosfopiruvato Hidratasa/metabolismo , Fosforilación , Sus scrofa , Espectrometría de Masas en Tándem , Extractos de Tejidos , Cadena B de alfa-Cristalina/química , Cadena B de alfa-Cristalina/metabolismoRESUMEN
Alkylhydroperoxide reductase (AhpC) is an abundant and important antioxidant protein present in Helicobacter pylori (HP), a spiral Gram-negative microaerophilic bacterium. By sequence alignment and structure comparison, HP-AhpC was found to be more homologous to human peroxiredoxins (hPrx) than to other eubacterial AhpC proteins. Similar to hPrxI, native HP-AhpC existed as a dimer of single subunit, comprising alpha-helix and beta-sheet domains with low surface hydrophobicity. AhpC can form high-molecular-weight (HMW) aggregates ranging from 700 to higher than 2,000 kDa under oxidative stress, possessing chaperone activity in the presence of thioredoxin (Trx). Further analysis of peroxide-reductase activities showed that HP-AhpC was more resistant to H(2)O(2) than hPrxI. However, the mechanism of enzyme inactivation to H(2)O(2) appeared to be similar for both HP-AhpC and hPrxI as revealed by native gel electrophoresis followed by proteomic identification using two-dimensional gel electrophoresis (2-DE) and LC-MS/MS. In contrast to T90D-hPrxI mutant with chaperone activity, site-specific mutant T87D-HP-AhpC did not form HMW chaperone complexes. The comparison of these two evolutionarily distant and yet functionally related enzymes may shed some light on the mechanism(s) underlying the evolution and development of the dual functionality in HP-AhpC and hPrxI with similar protein structure.
Asunto(s)
Helicobacter pylori/enzimología , Mutagénesis Sitio-Dirigida , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Secuencia de Aminoácidos , Cromatografía Liquida , Clonación Molecular , Electroforesis en Gel Bidimensional , Helicobacter pylori/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Peroxirredoxinas/química , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Espectrometría de Masas en TándemRESUMEN
Helicobacter pylori is a spiral Gram-negative microaerophilic bacterium. It is unique and distinctive among various bacterial pathogens for its ability to persist in the extreme acidic environment of human stomachs. To address and identify changes in the proteome of H. pylori in response to low pH, we have used a proteomic approach to study the protein expression of H. pylori under neutral (pH 7) and acidic (pH 5) conditions. Global protein-expression profiles of H. pylori under acid stress were analysed by two-dimensional polyacrylamide gel electrophoresis (2-DE) followed by liquid chromatography (LC)-nanoESI-mass spectrometry (MS)/MS and bioinformatics database analysis. Among the proteins differentially expressed under acidic condition, a non-heme iron-containing ferritin of H. pylori (HP-ferritin) was found to be consistently upregulated at pH 5 as compared to pH 7. It was also found that HP-ferritin can switch from an iron-storage protein with ferroxidase activity to a DNA-binding/protection function under in vitro conditions upon exposure to acidic environment. Prokaryotic ferritins, such as non-heme iron-binding HP-ferritin with dual functionality reported herein, may play a significant urease-independent role in the acid adaptation of H. pylori under physiological conditions in vivo.
Asunto(s)
Ceruloplasmina/metabolismo , Proteínas de Unión al ADN/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Helicobacter pylori/enzimología , Estrés Fisiológico/genética , Regulación hacia Arriba , Adaptación Fisiológica , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Ceruloplasmina/química , Ceruloplasmina/genética , Ceruloplasmina/aislamiento & purificación , Biología Computacional/métodos , Daño del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Regulación hacia Abajo , Ferritinas/química , Ferritinas/aislamiento & purificación , Perfilación de la Expresión Génica , Genes Bacterianos , Helicobacter pylori/genética , Helicobacter pylori/fisiología , Concentración de Iones de Hidrógeno , Microquímica/métodos , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , Proteómica/métodos , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Factores de TiempoRESUMEN
One novel snake venom factor (termed trimecetin) was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus). The purified venom factor was shown to consist of two subunit chains linked by one disulfide bond. This two-chain factor showed high sequence homology at their N-terminal segments to some previously reported venom proteins such as alboaggregin-B isolated from Trimeresurus albolabris and agkicetin from Agkistrodon acutus. The cDNA clones corresponding to the two subunit chains, a basic chain (pI 8.97) of 133 amino acids and an acidic chain (pI 6.32) of 121 amino acids, were found to share a sequence similarity of 42.6 %. Similar to botrocetin, bitiscetin and flavocetin A characterized from other snake species, trimecetin from Taiwan habu was also shown to be a C-type lectin based on the phylogenetic and sequence comparisons of various two-chain factors from snake species of different families. The unique functional variation and evolution of trimecetin may offer some insights into the mechanism underlying the receptor recognition associated with activation or inhibition of platelet aggregation for this family of snake venom proteins.
Asunto(s)
Venenos de Crotálidos/química , Lectinas Tipo C/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Humanos , Lectinas Tipo C/genética , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Conejos , Homología de Secuencia de Aminoácido , TrimeresurusRESUMEN
Using Caenorhabditis elegans as a model organism, various natural substances and commercial health-food supplements were screened to evaluate their effects on longevity. Among the substances tested, acetic acid and Reishi polysaccharide fraction 3 (RF3) were shown to increase the expression of the lifespan and longevity-related transcription factor DAF-16 in C. elegans. We have shown that RF3 activates DAF-16 expression via TIR-1 receptor and MAPK pathway whereas acetic acid inhibits the trans-membrane receptor DAF-2 of the insulin/IGF-1 pathway to indirectly activate DAF-16 expression. In addition, a mixture of acetic acid and RF3 possesses a combined effect 30-40% greater than either substance used alone. A proteomic analysis of C. elegans using 2-DE and LC-MS/MS was then carried out, and 15 differentially expressed proteins involved in the lifespan-promoting activity were identified.
Asunto(s)
Ácido Acético/farmacología , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/fisiología , Longevidad/efectos de los fármacos , Longevidad/fisiología , Polisacáridos/farmacología , Reishi/química , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Factores de Transcripción Forkhead , Regulación de la Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Longevidad/genética , Proteómica , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Espectrometría de Masas en Tándem , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: The COOH-terminal extension segment of alphaB-crystallin, a member of small heat shock protein (sHSP) family, appears to be a flexible polypeptide segment susceptible to proteolytic truncation and modifications under physiological conditions. To investigate its role on the structure and chaperone-like activity, we constructed various mutants of porcine alphaB-crystallin with either COOH-terminal serial truncations or site-specific mutagenesis on the last two residues. METHODS: The structures of these mutants were analyzed by circular dichroism (CD) spectroscopy, fluorescence spectra, mass spectrometry, Gel-permeation FPLC, and dynamic light-scattering spectrophotometry. Chaperone activity assays were performed under thermal and non-thermal stresses. The stability of proteins was examined by turbidity assays and CD spectra. RESULTS: All mutants showed similar secondary and tertiary structural features to the wild-type alphaB-crystallin as revealed by circular dichroism. However, truncations of the COOH-terminal segment generated crystallin aggregates with a molecular size slightly smaller than that of the wild-type alphaB-crystallin. The deletion of 12 residues from the COOH-terminal end greatly reduced the solubility, thermostability, and chaperone activity of alphaB-crystallin. On the contrary, the truncation of only 10 residues or less resulted in increased thermostability and enhanced anti-aggregation chaperone activity of alphaB-crystallin, with a maximal effect occurring on elimination of the last two residues. Moreover, displacing the last two lysines with glutamates or other neutral amino acids tended to show even higher chaperone activity than the deletion mutants. CONCLUSIONS: Our study clearly demonstrated that both the length and electrostatic charge of the COOH-terminal segment play crucial roles in governing the structural stability and chaperone activity of alphaB-crystallin.