RESUMEN
Gastric cancer is the second leading cause of cancer-related deaths worldwide. Gastric cancer is often detected at a late stage when treatment is difficult. Biomarkers for early detection and drug targets for gastric cancer therapy are critical for effective management of gastric cancer. Secreted proteins not only play integral roles in cancer progression and metastasis, they are also easily accessible. Secreted proteins within the tumor microenvironment are therefore an attractive source of biomarkers and drug targets. In this study, iTRAQ-based liquid chromatography/tandem mass spectrometry was used for comparative profiling of the secretomes of 11 gastric cancer cell lines versus a normal gastric epithelial cell line. Of the close to 800 proteins detected, about 600 proteins were detected to display differential expression in one or more gastric cancer cell lines compared to normal cells. These differentially expressed proteins predominantly have binding or enzymatic activities and are largely associated with cellular and metabolic processes. Overexpression of ARPC4 was validated in gastric cell lines and its novel function in gastric cancer cell migration and invasion demonstrated in vitro. The findings support the notion of ARPC4 as a potential biomarker/drug target for metastatic gastric cancer.
Asunto(s)
Proteínas de Neoplasias/metabolismo , Proteoma/metabolismo , Neoplasias Gástricas/metabolismo , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Movimiento Celular/fisiología , Perfilación de la Expresión Génica/métodos , Humanos , Invasividad Neoplásica/patología , Proteómica/métodos , Microambiente Tumoral/fisiologíaRESUMEN
Secreted proteins are an attractive minefield for cancer drug targets. An iTRAQ-based tandem mass spectrometry approach was employed to relatively quantify proteins in the secretomes of four isogenic breast cancer cell lines with increasing metastatic potential. CXCL3 was found to be upregulated in aggressive cancer cells. SiRNA and antibody neutralization studies supported a role of CXCL3 in metastatic processes. Meta-analysis of the mRNA level of CXCL3 in 1881 breast tumors supported a role of CXCL3 in clinical breast cancer. Our results support a functional role of CXCL3 in breast cancer metastasis and as a viable target for cancer therapy.
Asunto(s)
Neoplasias de la Mama/patología , Quimiocinas CXC/antagonistas & inhibidores , Metástasis de la Neoplasia , Neoplasias de la Mama/metabolismo , Quimiocinas CXC/genética , Quimiocinas CXC/fisiología , Femenino , Humanos , ARN Mensajero/genéticaRESUMEN
Phosphorylation of protein plays a key role in the regulation of cellular signal transduction and gene expression. In recent years, targeted mass spectrometry facilitates functional phosphoproteomics by allowing specific protein modifications of target proteins in complex samples to be characterized. In this study, we employed multiple reaction monitoring (MRM) to examine the influence of gefitinib (also known as Iressa) on the phosphorylation sites of EGFR protein before and after EGF treatment. By coupling MRM to MS/MS, 5 phosphotyrosine (Y1110, Y1172, Y1197, Y1069, and Y1092) and 1 S/T (T693) sites were identified on EGFR. Y1197 and T693 were constitutively phosphorylated. All phosphorylation sites were sensitive to gefitinib treatment except T693. Interestingly, gefitinib treatment induced phosphorylation of S1166 only in the presence of EGF. We further showed that lung cancer cells overexpressing phosphomimic S1166D EGFR mutant possessed significantly lower growth and proliferation property compared to wildtype EGFR-expressing cells. While the function and mode of regulation of S1166 remain unclear, our data supports the notion that S1166 represents a regulatory site that exerts a negative regulation on growth and proliferation of cancer cells. The data presented has implication in our understanding of dynamic drug (gefitinib)-target (EGFR) interaction and in improving the efficacy of target-directed therapeutics.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/fisiología , Receptores ErbB/metabolismo , Procesamiento Proteico-Postraduccional , Quinazolinas/farmacología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/química , Gefitinib , Humanos , Neoplasias Pulmonares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fosforilación , Serina/metabolismo , Espectrometría de Masas en Tándem , Tirosina/metabolismoRESUMEN
Cancer progression is governed by multifaceted interactions of cancer cells with their microenvironment and one of these ways is through secreted compounds. Substances released by gastric cancer cells have not being profiled in a proteome-wide manner. ITRAQ-based tandem mass spectrometry was employed to quantify proteins secreted by HFE145 normal, MKN7 well-differentiated, and MKN45 poorly differentiated gastric cancer cell lines. The expression levels of 237 proteins were found to be significantly different between normal and cancer cells. Further examination of 16 gastric cell lines and 115 clinical samples validated the up-regulation of CTSS expression in gastric cancer. Silencing CTSS expression suppressed the migration and invasion of gastric cancer cells in vitro. Subsequent secretomics revealed that CTSS silencing resulted in changes in expression levels of 197 proteins, one-third of which are implicated in cellular movement. Proteome-wide comparative secretomes of normal and gastric cancer cells were produced that constitute a useful resource for gastric cancer research. CTSS was demonstrated to play novel roles in gastric cancer cell migration and invasion, putatively via a network of proteins associated with cell migration, invasion, or metastasis. Cathepsin S is member of a large group of extracellular proteases, which are attractive drug targets. The implicated role of CTSS in gastric cancer metastasis provides an opportunity to test existing compounds against CTSS for adjuvant therapy and/or treatment of metastatic gastric cancers.
Asunto(s)
Catepsinas/metabolismo , Movimiento Celular/fisiología , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Catepsinas/química , Línea Celular Tumoral , Humanos , Marcaje Isotópico , Invasividad Neoplásica , Proteínas de Neoplasias/química , Proteómica/métodos , Reproducibilidad de los Resultados , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
Gastric cancer is one of the leading causes of cancer-related deaths worldwide. Current biomarkers used in the clinic do not have sufficient sensitivity for gastric cancer detection. To discover new and better biomarkers, protein profiling on plasma samples from 25 normal, 15 early-stage and 21 late-stage cancer was performed using an iTRAQ-LC-MS/MS approach. The level of C9 protein was found to be significantly higher in gastric cancer compared with normal subjects. Immunoblotting data revealed a congruent trend with iTRAQ results. The discriminatory power of C9 between normal and cancer states was not due to inter-patient variations and was independent from gastritis and Helicobacter pylori status of the patients. C9 overexpression could also be detected in a panel of gastric cancer cell lines and their conditioned media compared with normal cells, implying that higher C9 levels in plasma of cancer patients could be attributed to the presence of gastric tumor. A subsequent blind test study on a total of 119 plasma samples showed that the sensitivity of C9 could be as high as 90% at a specificity of 74%. Hence, C9 is a potentially useful biomarker for gastric cancer detection.
Asunto(s)
Biomarcadores de Tumor/sangre , Complemento C9/metabolismo , Neoplasias Gástricas/sangre , Regulación hacia Arriba , Anciano , Línea Celular Tumoral , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Gastric cancer has one of the highest morbidities and mortalities worldwide. Early detection is key measure to improve the outcome of gastric cancer patients. In our efforts to identify potential markers for gastric cancer detection, we coupled xenotransplantation mouse model with a plasma proteomic approach. MKN45 gastric cancer cells were subcutaneously injected into nude mice and plasma samples from mice bearing different sizes of tumors were collected and subjected to iTRAQ and mass spectrometry analysis. ITIH3 protein was found to be more highly expressed in plasma of tumor bearing mice compared to control. Subsequent screening of ITIH3 expression in 167 clinical plasma samples, including 83 cancer-free subjects and 84 gastric cancer patients, revealed higher ITIH3 level in the plasma of gastric cancer patients. A receiver operating characteristics (ROC) curve estimated a maximal sensitivity of 96% at 66% specificity for ITIH3 in gastric cancer detection. In addition, plasma from early stage gastric cancer patient has significantly (p < 0.001) higher level of ITIH3 compared to that from noncancer subject. Our data suggest that ITIH3 may be a useful biomarker for early detection of gastric cancer.
Asunto(s)
alfa-Globulinas/metabolismo , Biomarcadores de Tumor/metabolismo , Proteoma/metabolismo , Neoplasias Gástricas/metabolismo , alfa-Globulinas/análisis , Animales , Biomarcadores de Tumor/análisis , Proteínas Sanguíneas/análisis , Western Blotting , Línea Celular Tumoral , Densitometría , Histocitoquímica , Humanos , Marcaje Isotópico , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Proteoma/análisis , Curva ROC , Reproducibilidad de los Resultados , Neoplasias Gástricas/diagnóstico , Trasplante HeterólogoRESUMEN
BACKGROUND: Mapping the expression changes during breast cancer development should facilitate basic and translational research that will eventually improve our understanding and clinical management of cancer. However, most studies in this area are challenged by genetic and environmental heterogeneities associated with cancer. METHODOLOGY/PRINCIPAL FINDINGS: We conducted proteomics of the MCF10AT breast cancer model, which comprises of 4 isogenic xenograft-derived human cell lines that mimic different stages of breast cancer progression, using iTRAQ-based tandem mass spectrometry. Of more than 1200 proteins detected, 98 proteins representing at least 20 molecular function groups including kinases, proteases, adhesion, calcium binding and cytoskeletal proteins were found to display significant expression changes across the MCF10AT model. The number of proteins that showed different expression levels increased as disease progressed from AT1k pre-neoplastic cells to low grade CA1h cancer cells and high grade cancer cells. Bioinformatics revealed that MCF10AT model of breast cancer progression is associated with a major re-programming in metabolism, one of the first identified biochemical hallmarks of tumor cells (the "Warburg effect"). Aberrant expression of 3 novel breast cancer-associated proteins namely AK1, ATOX1 and HIST1H2BM were subsequently validated via immunoblotting of the MCF10AT model and immunohistochemistry of progressive clinical breast cancer lesions. CONCLUSION/SIGNIFICANCE: The information generated by this study should serve as a useful reference for future basic and translational cancer research. Dysregulation of ATOX1, AK1 and HIST1HB2M could be detected as early as the pre-neoplastic stage. The findings have implications on early detection and stratification of patients for adjuvant therapy.
Asunto(s)
Neoplasias de la Mama/patología , Modelos Biológicos , Proteínas de Neoplasias/genética , Proteoma , Neoplasias de la Mama/genética , Línea Celular Tumoral , Cromatografía Liquida , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Espectrometría de Masas en TándemRESUMEN
There is no suitable diagnostic and prognostic biomarker for gastric cancer. The biggest hurdles in biomarker discovery are (i) the low abundance of cancer cell-specific proteins that limits their detection and (ii) complex inter-patient variations that complicate the discovery process. To circumvent these issues, we conducted proteomics on the plasma of gastric cancer mouse xenograft and attempted to identify proteins released by cancer cells. MKN45 gastric cancer cells were subcutaneously implanted into immune-incompetent nude mice. Plasma samples collected from mice with different tumor sizes (low, mid and high tumor loads) were subjected to iTRAQ and mass spectrometric analyses. Detection of human APOA1 in mouse plasma was verified and its expression level was shown to be lower in mice with large tumors compared to those with small tumors. Studies on a panel of about 14 gastric cancer cell lines supported the notion that APOA1 in mouse plasma was of human gastric cancer cell origin. While the clinical utility of APOA1 remains to be ascertained with a larger scale study, the current work supported the feasibility of using mouse xenograft model for gastric cancer biomarker discovery.
Asunto(s)
Apolipoproteína A-I/sangre , Biomarcadores de Tumor/sangre , Neoplasias Gástricas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Datos de Secuencia Molecular , Trasplante de Neoplasias , Proteómica/métodos , Alineación de Secuencia , Neoplasias Gástricas/genéticaRESUMEN
With the use of the breast cancer metastatic model, which comprises four isogenic cell lines, iTRAQ-based ESI-LC/MS/MS proteomics was employed to catalog protein expression changes as cancer cells acquire increasing metastatic potential. From more than 1000 proteins detected, 197 proteins, including drug-targetable kinases, phosphatases, proteases and transcription factors, displayed differential expression when cancer cells becomes more metastatic. Overall, the number of protein expression changes was evenly distributed across mildly ( approximately 30%), moderately ( approximately 40%) and aggressively ( approximately 30%) metastatic cancer cells. Some changes were found to be specific to one while others were required for two or more phenotypes. KEGG Orthology suggests major reprogramming in cell metabolism and to smaller extents in genetic and environmental information processing. Ten novel metastasis-associated proteins were identified and the iTRAQ-based expression profiles of 7 proteins were verified to be congruent with antibody-based methods. With the use of tissue microarrays comprising 50 matched cases of invasive and metastatic lesions, the expression profiles of SH3GLB1 and SUB1, SND1, TRIM28 were validated to be down- and up-regulated, respectively, during clinical progression of carcinoma in situ to invasive and metastatic carcinomas. Our study has unraveled proteome-wide molecular aberrations and potentially new players in breast cancer metastasis.
Asunto(s)
Neoplasias de la Mama/química , Neoplasias de la Mama/patología , Metástasis de la Neoplasia , Proteínas de Neoplasias/química , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Análisis por Matrices de Proteínas/métodos , Reproducibilidad de los Resultados , Análisis de Matrices TisularesRESUMEN
The past 5 years have seen an explosion of phosphoproteomics methods development. In this review, using epidermal growth-factor signaling as a model, we will discuss how phosphoproteomics, along with bioinformatics and computational modeling, have impacted key aspects of oncogenic signaling such as in the temporal fine mapping of phosphorylation events, and the identification of novel tyrosine kinase substrates and phosphorylation sites. We submit that the next decade will see considerable exploitation of phosphoproteomics in cancer research. Such a phenomenon is already happening as exemplified by its use in promoting the understanding of the molecular etiology of cancer and target-directed therapeutics.
Asunto(s)
Neoplasias/metabolismo , Oncogenes/fisiología , Fosfoproteínas/fisiología , Proteómica/métodos , Animales , Transformación Celular Neoplásica/metabolismo , Biología Computacional , Simulación por Computador , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/fisiología , Receptores ErbB/genética , Receptores ErbB/fisiología , Humanos , Neoplasias/genética , Fosforilación , Transducción de SeñalRESUMEN
We have identified and characterized the proteome of Sulfolobus solfataricus P2 using multidimensional liquid phase protein separations. Multidimensional liquid phase chromatography was performed using ion exchange chromatography in the first dimension, followed by reverse-phase chromatography using 500 microm i.d. poly(styrene-divinylbenzene) monoliths in the second dimension to separate soluble protein lysates from S. solfataricus. The 2DLC protein separations from S. solfataricus protein lysates enabled the generation of a 2D liquid phase map analogous to the traditional 2DE map. Following separation of the proteins in the second dimension, fractions were collected, digested in solution using trypsin and analyzed using mass spectrometry. These approaches offer significant reductions in labor intensity and the overall time taken to analyze the proteome in comparison to 2DE, taking advantage of automation and fraction collection associated with this approach. Furthermore, following proteomic analysis using 2DLC, the data obtained was compared to previous 2DE and shotgun proteomic studies of a soluble protein lysate from S. solfataricus. In comparison to 2DE, the results show an overall increase in proteome coverage. Moreover, 2DLC showed increased coverage of a number of protein subsets including acidic, basic, low abundance and small molecular weight proteins in comparison to 2DE. In comparison to shotgun studies, an increase in proteome coverage was also observed. Furthermore, 187 unique proteins were identified using 2DLC, demonstrating this methodology as an alternative approach for proteomic studies or in combination with 2DE and shotgun workflows for global proteomics.
Asunto(s)
Proteínas Arqueales/análisis , Proteoma/análisis , Sulfolobus solfataricus/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Cromatografía Liquida/métodos , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/aislamiento & purificación , Punto Isoeléctrico , Metilación , Peso Molecular , Procesamiento Proteico-Postraduccional , Proteoma/química , Proteoma/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Tripsina/químicaRESUMEN
To identify novel tyrosine kinase substrates that have never been implicated in cancer, we studied the phosphoproteomic changes in the MCF10AT model of breast cancer progression using a combination of phosphotyrosyl affinity enrichment, iTRAQ technology, and LC-MS/MS. Using complementary MALDI- and ESI-based mass spectrometry, 57 unique proteins comprising tyrosine kinases, phosphatases, and other signaling proteins were detected to undergo differential phosphorylation during disease progression. Seven of these proteins (SPAG9, Toll-interacting protein (TOLLIP), WBP2, NSFL1C, SLC4A7, CYFIP1, and RPS2) were validated to be novel tyrosine kinase substrates. SPAG9, TOLLIP, WBP2, and NSFL1C were further proven to be authentic targets of epidermal growth factor signaling and Iressa (gefitinib). A closer examination revealed that the expression of SLC4A7, a bicarbonate transporter, was down-regulated in 64% of the 25 matched normal and tumor clinical samples. The expression of TOLLIP in clinical breast cancers was heterogeneous with 25% showing higher expression in tumor compared with normal tissues and 35% showing the reverse trend. Preliminary studies on SPAG9, on the other hand, did not show differential expression between normal and diseased states. This is the first time SLC4A7 and TOLLIP have been discovered as novel tyrosine kinase substrates that are also associated with human cancer development. Future molecular and functional studies will provide novel insights into the roles of TOLLIP and SLC4A7 in the molecular etiology of breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Proteínas Tirosina Quinasas/metabolismo , Neoplasias de la Mama/enzimología , Línea Celular Tumoral , Cromatografía Liquida , Humanos , Inmunohistoquímica , Fosforilación , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato , Espectrometría de Masas en TándemRESUMEN
Sulfolobus solfataricus P2 was shown to survive on ethanol at various concentrations (0.08-3.97% w/v) as the sole carbon source. The highest ethanol consumption rate was 15.1 mg/L/hr (via GC-MS analysis) in cultures grown on 0.79% w/v ethanol. In vivo metabolic labeling, using 13C universally labeled ethanol, provided evidence for both ethanol uptake and metabolic utilization. Results obtained from isobaric mass tag-facilitated shotgun proteomics (iTRAQ) indicate that on average, 21 and 31% of the 284 proteins identified (with > or = 2 MS/MS) are increased and decreased expression in ethanol cultures compared to glucose control cultures. Preliminary analysis shows >2-fold increase of the zinc-dependent alcohol dehydrogenase, ADH-10 (SSO2536), and the putative ADH-2 (SSO0764) in both translational and transcriptional data (using quantitative RT-PCR), suggesting both proteins are integral to ethanol metabolism. Evidence that ethanol was catabolised into central metabolism via acetyl-CoA intermediates was further indicated by another >2-fold increase in protein expression levels of various acetyl-CoA synthetases. The decreased expression (>2-fold) of isocitrate dehydrogenase at the protein level suggests that the ethanol grown cultures shifted toward the glyoxylate cycle. Subsequently, the activity of ADH-2 was confirmed by overexpression in Escherichia coli, with the resultant purified in vitro enzyme exhibiting an activity that increased with temperature up to 95 degrees C, and giving a specific activity of 1.05 U/mg.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Proteínas Arqueales/metabolismo , Etanol/metabolismo , Proteoma/metabolismo , Sulfolobus solfataricus/metabolismo , Alcohol Deshidrogenasa/biosíntesis , Alcohol Deshidrogenasa/genética , Proteínas Arqueales/genética , Medios de Cultivo , Escherichia coli/enzimología , Cinética , Proteoma/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfolobus solfataricus/enzimología , Sulfolobus solfataricus/fisiología , Espectrometría de Masas en Tándem , Transcripción GenéticaRESUMEN
Sulfolobus solfataricus P2 is able to metabolize n-propanol as the sole carbon source. An average n-propanol consumption rate of 9.7 and 3.3 mg/L/hr was detected using GC-MS analysis from S. solfataricus cultures grown in 0.40 and 0.16% w/v n-propanol, respectively. The detection of propionaldehyde, the key intermediate of n-propanol degradation, produced at a rate of 1.3 and 1.0 mg/L/hr in 0.40 and 0.16% w/v n-propanol cultures, further validated the ability of S. solfataricus to utilize n-propanol. The translational and transcriptional responses of S. solfataricus grown on n-propanol versus glucose were also investigated using quantitative RT-PCR and iTRAQ approaches. Approximately 257 proteins with > or =2 MS/MS spectra were identified and quantified via iTRAQ. The global quantitative proteome overview obtained showed significant up-regulation of acetyl-CoA synthetases, propionyl-CoA carboxylase, and methylmalonyl-CoA mutase enzymes. This led to the proposition that the propionyl-CoA formed from n-propanol degradation is catabolised into the citrate cycle (central metabolism) via succinyl-CoA intermediates. In contrast, evidence obtained from these analysis approaches and in vivo stable isotope labeling experiments, suggests that S. solfataricus is only capable of converting isopropyl alcohol to acetone (and vice versa) but lacks the ability to further metabolize these compounds.
Asunto(s)
1-Propanol/metabolismo , Proteoma/análisis , Sulfolobus solfataricus/enzimología , Oxidorreductasas de Alcohol/análisis , Espectrometría de Masas , Biosíntesis de Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
We assess the reliability of isobaric-tags for relative and absolute quantitation (iTRAQ), based on different types of replicate analyses taking into account technical, experimental, and biological variations. In total, 10 iTRAQ experiments were analyzed across three domains of life involving Saccharomyces cerevisiae KAY446, Sulfolobus solfataricus P2, and Synechocystis sp. PCC 6803. The coverage of protein expression of iTRAQ analysis increases as the variation tolerance increases. In brief, a cutoff point at +/-50% variation (+/-0.50) would yield 88% coverage in quantification based on an analysis of biological replicates. Technical replicate analysis produces a higher coverage level of 95% at a lower cutoff point of +/-30% variation. Experimental or iTRAQ variations exhibit similar behavior as biological variations, which suggest that most of the measurable deviations come from biological variations. These findings underline the importance of replicate analysis as a validation tool and benchmarking technique in protein expression analysis.
Asunto(s)
Proteínas Arqueales/química , Proteínas Bacterianas/química , Proteómica/métodos , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/crecimiento & desarrollo , Sulfolobus solfataricus/crecimiento & desarrollo , Synechocystis/crecimiento & desarrollo , Proteínas Arqueales/aislamiento & purificación , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Indicadores y Reactivos , Espectrometría de Masas/métodos , Reproducibilidad de los Resultados , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The potential of Sulfolobus solfataricus P2 for alcohol or ketone bioconversion was explored in this study. S. solfataricus was grown in different concentrations (0.1-0.8% w/v) of alcohols or ketones (ethanol, iso-propanol, n-propanol, acetone, phenol and hexanol) in the presence of 0.4% w/v glucose. Consequently, the addition of these alcohols or ketones into the growth media had an inhibitory effect on biomass production, whereby lag times increased and specific growth rates decreased when compared to a glucose control. Complete glucose utilisation was observed in all cultures, although slower rates of glucose consumption were observed in experimental cultures (average of 14.9 mg/L/h compared to 18.9 mg/L/h in the control). On the other hand, incomplete solvent utilisation was observed, with the highest solvent consumption being approximately 51% of the initial concentration in acetone cultures. Translational responses of S. solfataricus towards these alcohols or ketones were then investigated using the isobaric tags for relative and absolute quantitation (iTRAQ) technique. The majority (>80%) of proteins identified and quantified showed no discernable changes in regulation compared to the control. These results, along with those obtained from transcriptional analysis of key genes involved within this catabolic process using quantitative RT-PCR and metabolite analysis, demonstrate successful alcohol or ketone conversion in S. solfataricus.
Asunto(s)
Alcoholes/metabolismo , Perfilación de la Expresión Génica , Cetonas/metabolismo , Sulfolobus solfataricus/genética , Biosíntesis de Proteínas/fisiología , Sulfolobus solfataricus/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Saccharomyces cerevisiae KAY446 was utilized for ethanol production, with glucose concentrations ranging from 120 g/L (normal) to 300 g/L (high). Although grown in a high glucose environment, S. cerevisiae still retained the ability to produce ethanol with a high degree of glucose utilization. iTRAQ-mediated shotgun proteomics was applied to identify relative expression change of proteins under the different glucose conditions. A total of 413 proteins were identified from three replicate, independent LC-MS/MS runs. Unsurprisingly, many proteins in the glycolysis/gluconeogenesis pathway showed significant changes in expression level. Twenty five proteins involved in amino acid metabolism decreased their expression, while the expressions of 12 heat-shock related proteins were also identified. Under high glucose conditions, ethanol was produced as a major product. However, the assimilation of glucose as well as a number of byproducts was also enhanced. Therefore, to optimize the ethanol production under very high gravity conditions, a number of pathways will need to be deactivated, while still maintaining the correct cellular redox or osmotic state. Proteomics is demonstrated here as a tool to aid in this forward metabolic engineering.
Asunto(s)
Etanol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucólisis/genética , Hipergravedad , Proteómica , Saccharomyces cerevisiae , Fermentación , Perfilación de la Expresión Génica , Glucosa , Espectrometría de MasasRESUMEN
Cyanobacteria are photosynthetic bacteria notable for their ability to produce hydrogen and a variety of interesting secondary metabolites. As a result of the growing number of completed cyanobacterial genome projects, the development of post-genomics analysis for this important group has been accelerating. DNA microarrays and classical two-dimensional gel electrophoresis (2DE) were the first technologies applied in such analyses. In many other systems, 'shotgun' proteomics employing multi-dimensional liquid chromatography and tandem mass spectrometry has proven to be a powerful tool. However, this approach has been relatively under-utilized in cyanobacteria. This study assesses progress in cyanobacterial shotgun proteomics to date, and adds a new perspective by developing a protocol for the shotgun proteomic analysis of the filamentous cyanobacterium Anabaena variabilis ATCC 29413, a model for N(2) fixation. Using approaches for enhanced protein extraction, 646 proteins were identified, which is more than double the previous results obtained using 2DE. Notably, the improved extraction method and shotgun approach resulted in a significantly higher representation of basic and hydrophobic proteins. The use of protein bioinformatics tools to further mine these shotgun data is illustrated through the application of PSORTb for localization, the grand average hydropathy (GRAVY) index for hydrophobicity, LipoP for lipoproteins and the exponentially modified protein abundance index (emPAI) for abundance. The results are compared with the most well-studied cyanobacterium, Synechocystis sp. PCC 6803. Some general issues in shotgun proteome identification and quantification are then addressed.
Asunto(s)
Cianobacterias/química , Proteoma/análisis , Proteómica/métodos , Biología ComputacionalRESUMEN
We analyzed 10 isobaric tags for relative and absolute quantitation (iTRAQ) experiments using three different model organisms across the domains of life: Saccharomyces cerevisiae KAY446, Sulfolobussolfataricus P2, and Synechocystis sp. PCC6803. A double database search strategy was employed to minimize the rate of false positives to less than 3% for all organisms. The reliability of proteins with single-peptide identification was also assessed using the search strategy, coupled with multiple analyses of samples into LC-MS/MS. The outcomes of the three LC-MS/MS analyses provided higher proteome coverage with an average increment in total proteins identified of 6%, 33%, and 50% found in S. cerevisiae, S. solfataricus, and Synechocystis sp., respectively. The iTRAQ quantification values were found to be highly reproducible across the injections, with an average coefficient of variation (CV) of 0.09 (scattering from 0.14 to 0.04) calculated based on log mean average ratio for all three organisms. Hence, we recommend multiple analyses of iTRAQ samples for greater proteome coverage and precise quantification.
Asunto(s)
Proteínas/análisis , Proteómica/métodos , Secuencia de Aminoácidos , Cromatografía Liquida , Bases de Datos de Proteínas , Reacciones Falso Positivas , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Reproducibilidad de los Resultados , Proteínas de Saccharomyces cerevisiae/química , Sulfolobus solfataricus/química , Synechocystis/químicaRESUMEN
Via combined separation approaches, a total of 1399 proteins were identified, representing 47% of the Sulfolobus solfataricus P2 theoretical proteome. This includes 1323 proteins from the soluble fraction, 44 from the insoluble fraction and 32 from the extra-cellular or secreted fraction. We used conventional 2-dimensional gel electrophoresis (2-DE) for the soluble fraction, and shotgun proteomics for all three cell fractions (soluble, insoluble, and secreted). Two gel-based fractionation methods were explored for shotgun proteomics, namely: (i) protein separation utilizing 1-dimensional gel electrophoresis (1-DE) followed by peptide fractionation by iso-electric focusing (IEF), and (ii) protein and peptide fractionation both employing IEF. Results indicate that a 1D-IEF fractionation workflow with three replicate mass spectrometric analyses gave the best overall result for soluble protein identification. A greater than 50% increment in protein identification was achieved with three injections using LC-ESI-MS/MS. Protein and peptide fractionation efficiency; together with the filtration criteria are also discussed.