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ABSTRACT: Fungal species of the genus Aspergillus fumigatus ubiquitous saprophytes that play major role in lignocellulosic biomass recycling and also considered as cell factories for the production of organic acids, pharmaceuticals and industrially important enzymes. Analysis of extracellular secreted biomass degrading enzymes using complex lignocellulosic biomass as a substrate by solid-state fermentation could be more practical approach to evaluate application of the enzymes for lignocellulosic biorefinery. This study isolated fungal strain from compost, identified as Aspergillus Fumigatus and further analyzed it for lignocellulolytic enzymes at different temperatures using label free quantitative proteomics. The profile of secretome composition discovered cellulases, hemicellulases, lignin degrading proteins, peptidases and proteases, transport and hypothetical proteins; while protein abundances and further their hierarchical clustering analysis revealed temperature dependent expression of these enzymes during solid state fermentation of saw dust. The enzyme activities and protein abundances as determined by exponentially modified protein abundance index (emPAI) indicated the maximum activities at the range of 40-50 ºC, demonstrating thermophilic nature of the isolate A. fumigatus LF9. Characterization of thermostability of secretome suggested the potential of the isolated fungal strain in the production of thermophilic biomass degrading enzymes for industrial application.
Journal of Proteome Research 05/2013; · 5.11 Impact Factor
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ABSTRACT: POPX2 is a serine/threonine phosphatase belonging to the protein phosphatase 2C (PP2C) family that has been found to be elevated in invasive breast cancer cells. Silencing of POPX2 results in lower cell motility and invasiveness. The molecular mechanism of POPX2-regulated cell motility is not well understood. To identify the relevant signaling pathways, we investigated the transcriptome and proteome of POPX2-knockdown MDA-MB-231 breast cancer cells. Our data suggest that POPX2 might be involved in the regulation of focal adhesions and cytoskeleton dynamics through the regulation of MAP kinase (MAPK1/3) and glycogen synthase kinase 3 (GSK3α/β) activities. Silencing POPX2 alters phosphorylation levels of MAPK1/3 and GSK3α/β and results in reduced activity of these kinases. Both MAPK and GSK3 are known to regulate the activities of transcription factors. MAPK1/3 are also implicated in the phosphorylation of stathmin. The level of phospho-stathmin was found to be lower in POPX2 knockdown cells. As phosphorylation of stathmin inhibits its microtubule severing activity, we observed less stable microtubules in POPX2 knockdown cells. Taken together, our data suggest that POPX2 might regulate cell motility through its regulation of the MAPK1/3, leading to changes in the cytoskeleton and cell motility.
Journal of Proteome Research 04/2013; · 5.11 Impact Factor
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ABSTRACT: Protein abundance determination across multiple samples proved to be a daunting task and far fewer methods has been successfully devised for this purpose. Despite the technical challenges faced, protein abundance determination over multiple samples is still an area of interest. Herein, we introduce a new method for estimation of Protein Abundances in Multiplexed Samples (PAMUS). Protein abundance in the multiplexed sample comprising of the eight complex secretomes by Trichoderma reesei QM6a and Rut C30 grown in four different carbon sources, namely glucose, cellulose, starch, and a mixture of starch and cellulose were determined. For protein abundance in the multiplexed sample, Exponentially Modified Protein Abundance Index (emPAI) was used. Using the PAMUS method, we estimated the abundance of extracellular lignocellulolytic proteins secreted by two T. reesei strains in response to various carbon sources. The results reveal that cellulose induces biosynthesis of cellulases. PAMUS analysis of the secretomes implicates T. reesei Rut C30 as a hyper cellulolytic strain and further revealed the optimum concentrations of each secreted enzyme during cellulosic substrate utilization. Our study demonstrates the plausible use of PAMUS method for designing enzyme cocktails for optimum cellulose hydrolysis, and its potential applications in future studies involving other multiplexed biological samples. BIOLOGICAL SIGNIFICANCE: Relative protein quantitation across multiple complex biological samples dominates the field of quantitative proteomics. Protein abundance determination across multiple samples, on the other hand, proves to be a daunting task and far fewer methods have been successfully devised for this purpose. Despite the technical challenges faced, protein abundance determination over multiple samples is still an area of interest as it provides unique information about the biological processes, and physiological states of particular disease, or that of microbes. This study introduces a new method of estimation of protein abundance in multiplexed samples (PAMUS) which is applied to study eight complex secretomes by Trichoderma reesei QM6a and Rut C30 grown in different carbon sources. The expression level of proteins in the multiplexed eight complex secretome samples were determined by isobaric tags for relative and absolute quantitation (iTRAQ) reagents coupled with exponentially modified protein abundance index (emPAI) to calculate the abundance of each identified protein in eight conditions. This method with microbial secretome as an example facilitated a direct comparison of the abundance of proteins. This PAMUS could be applied for any multiple biological samples, for example, to study human disease to evaluate dynamic expression of proteins during disease progressions.
Journal of proteomics 04/2013; · 5.07 Impact Factor
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ABSTRACT: ERLIC and high-pH RP (Hp-RP) have been reported to be promising alternatives to strong cation exchange (SCX) in proteome fractionation. Here we compare the performance of ERLIC, concatenated ERLIC and concatenated Hp-RP in proteome profiling. The protein identification is comparable in these three strategies, but significantly more unique peptides are identified by the two concatenation methods, resulting in a significant increase of the average protein sequence coverage. The pooling of fractions from spaced intervals results in more uniform distribution of peptides in each fraction compared with the chromatogram-based pooling of adjacent fractions. ERLIC fractionated peptides according to their pI and GRAVY value. These properties remained but became less remarkable in concatenated ERLIC. In contrast, the average pI and GRAVY value of the peptides are comparable in each fraction in concatenated Hp-RP. ERLIC performs the best in identifying peptides with pI>9 among the three strategies, while concatenated Hp-RP was good at identifying peptides with pI<4. These advantages are useful when either basic or acidic peptides/proteins are analytical targets. The power of ERLIC in identification of basic peptides seems to be due to their efficient separation from acidic peptides. This study facilitates the choice of proper fractionation strategies based on specific objectives. SIGNIFICANCE: This work conducts a direct comparison of ERLIC, concatenated ERLIC and concatenated Hp-RP in fractionating rat kidney proteome. These three fractionation methods are comparable in identifying proteins, but concatenated ERLIC and concatenated Hp-RP identify significantly more unique peptides than ERLIC because the multiple fraction concatenation strategy results in the more efficient use of the separation space in the second-dimensional separation. Each method has some advantages. ERLIC is good at analyzing basic peptides due to their separation from acidic peptides, while concatenated Hp-RP performs the best in analyzing acidic peptides with pI <4. This will facilitate the choice of the proper peptide fractionation strategy based on a specific need. A combination of different fractionation strategies can be used to increase the sequence coverage and number of protein identification due to the complementary effect between different methods.
Journal of proteomics 02/2013; · 5.07 Impact Factor
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Danny A Kanhai,
Frank L J Visseren,
Yolanda van der Graaf,
Arjan H Schoneveld,
Louise M Catanzariti,
Leo Timmers,
L Jaap Kappelle,
Cuno S P M Uiterwaal,
Sai Kiang Lim, Siu Kwan Sze,
Gerard Pasterkamp,
Dominique P V de Kleijn
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ABSTRACT: BACKGROUND AND OBJECTIVES: Microvesicles (MVs) are small membrane vesicles that are involved in atherotrombotic processes. In the present study, we evaluated the risk of MV protein levels on the occurrence of new vascular events in patients with clinically manifest vascular disease. METHODS: In this cohort study 1060 patients were prospectively followed for the occurrence of a new vascular event or death (median follow up 6.4years, interquartile range 5.2-7.3years). MVs were isolated from plasma and MV protein levels of Cystatin C, Serpin G1, Serpin F2 and CD14 were measured. Multivariable Cox proportional hazards models were used to estimate the risk for new vascular events, vascular mortality and all-cause mortality. During follow up 136 vascular events occurred, 65 vascular mortality and 114 all-cause mortality. RESULTS: An increase in 1 standard deviation (SD) of Cystatin C MV level was related to an increased risk for myocardial infarction (HR 1.49; 95%CI 1.20-1.86), vascular mortality (HR 1.48; 95%CI 1.17-1.86), vascular events (HR 1.27; 1.07-1.52) and all-cause mortality (HR 1.41; 95%CI 1.18-1.69). Serpin F2 MV levels were related to an increased risk for myocardial infarction (HR 1.22; 95%CI 1.00-1.51), vascular mortality (HR 1.25; 95%CI 1.00-1.56), and all-cause mortality (HR 1.22; 95% CI 1.03-1.45). CD14 MV levels were related to an increased risk for myocardial infarction (HR 1.55; 95%CI 1.27-1.91), vascular mortality (HR 1.37; 95%CI 1.10-1.70), vascular events (HR 1.32; 95%CI 1.12-1.55), all-cause mortality (HR 1.36; 95%CI 1.15-1.62) and occurrence of ischemic stroke (HR 1.32; 95%CI 1.00-1.74). CONCLUSIONS: Cystatin C, Serpin F2 and CD14 MV levels are related to an elevated risk for future vascular events and mortality in patients with clinically manifest vascular disease.
International journal of cardiology 02/2013; · 7.08 Impact Factor
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Siu Kwan Sze,
Dominique P V de Kleijn,
Ruenn Chai Lai,
Eileen Khia Way Tan,
Hui Zhao,
Keng Suan Yeo,
Teck Yew Low,
Qizhou Lian,
Chuen Neng Lee,
Wayne Mitchell,
Reida Menshawe El Oakley,
Sai-Kiang Lim
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ABSTRACT: p60 transcription regulator protein (p60TRP) facilitates the processing of the amyloid precursor protein towards the non-amyloidogenic pathway by inhibiting the β-secretase action. This protein was initially identified to be downregulated in the temporal lobe of brains from Alzheimer's disease patients. p60TRP is one of the G-protein-coupled receptor (GPCR)-associated proteins which directly influences the signalling capacity of GPCRs. In the present study, we investigated the brain-region-specific proteome profile of transgenic p60TRP mice to gain an insight into the molecular events mediated by the long-term effect of neuronal p60TRP overexpression on brain proteome changes and its potential implication for neuronal functions in the central nervous system. Using a proteomics research approach based on isobaric tags for relative and absolute quantitation, we identified 2,025 proteins, whereby 1,735 proteins were quantified, out of which 56 were found to be significantly altered in the cortex and/or hippocampus of neuronal transgenic neuronal p60TRP mice. Our data suggests that in vivo overexpression of neuronal p60TRP significantly affects cognitive and neuroprotective capacities.
Neurosignals 02/2013; · 2.11 Impact Factor
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Vince C de Hoog,
Leo Timmers,
Arjan H Schoneveld,
Jiong-Wei Wang,
Sander M van de Weg, Siu Kwan Sze,
van Keulen,
Arno W Hoes,
Hester M den Ruijter,
Dominique PV de Kleijn,
Arend Mosterd
European Heart Journal: Acute Cardiovascular Care. 01/2013; 2(1):53-60.
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ABSTRACT: Tumor hypoxia induces cancer cell angiogenesis, invasiveness, treatment resistance, and contributes to poor clinical outcome. However, the molecular mechanism by which tumor hypoxia exerted a coordinated effect on different molecular pathways to enhance tumor growth and survival and lead to poor clinical outcome is not fully understood. In this study, we attempt to elucidate the global protein expression and functional changes in A431 epithelial carcinoma cells induced by hypoxia and reoxygenation using iTRAQ quantitative proteomics and biochemical functional assays. Quantitative proteomics results showed that 4,316 proteins were quantified with FDR<1%, in which over 1,200 proteins were modulated >1.2 folds, and DNA repair, glycolysis, integrin, glycoprotein turnover and STAT1 pathways were perturbed by hypoxia and reoxygenation-induced oxidative stress. For the first time, hypoxia was shown to up-regulate the nonhomologous end-joining (NHEJ) pathway which plays a central role in DNA repair of irradiated cells, thereby potentially contributing to the radioresistance of hypoxic A431 cells. The up-regulation of Ku70/Ku80 dimer, a key molecular complex in the NHEJ pathway, was confirmed by WB and LC-MS/MS-MRM methods. Functional studies confirmed that up-regulation of glycolysis, integrin, glycoprotein synthesis and down-regulation of STAT1 pathways during hypoxia enhanced metastastic activity of A431 cells. Migration of A431 cells was dramatically repressed by glycolysis inhibitor (2-Deoxy-D-glucose), glycoprotein synthesis inhibitor (1-Deoxynojirimycin Hydrochloride), and STAT1a overexpression that enhanced the integrin-mediated cell adhesion. These results revealed that hypoxia induced several biological processes involved in tumor migration and radioresistance and provided potential new targets for tumor therapy.
Molecular & Cellular Proteomics 11/2012; · 7.40 Impact Factor
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ABSTRACT: Proteomics analysis of lignocellulolytic proteins by lignocellulosic biomass degrading microbes and compatible microbial consortium is a promising approach that offers a new means to enzyme discovery. The abundance of proteins in complex secretome by microbial communities would highlight key lignocellulolytic proteins for lignocellulosic biorefinery. In this study, lignocellulolytic enzymes of potent lignin degrading basidiomycota and effective cellulolytic ascomycota fungal strains, and their co-cultures were analyzed using high throughput isobaric tag for relative and absolute quantitation (iTRAQ) technique using liquid chromatography tandem mass spectrometry. Protein abundances in the iTRAQ-multiplexed samples were determined by integrating relative quantitation and exponentially modified protein abundance index (emPAI). The functional classification of the secretory proteins by individual culture and co-culture demonstrated 36.77% cellulolytic proteins, 13.06% hemicellulases, 14.09% ligninolytic proteins, 19.59% proteolytic enzymes. 7.22% hypothetical proteins and 6.87% cell morphogenesis proteins. The abundance of the proteins by individual cultures and co-cultured fungal consortium revealed that co-culturing of Phanerochaete chrysosporium with Trichoderma reesei QM6a and Trichoderma reesei Rut C30 induced the production of cellulolytic proteins and stimulated expression of hemicellulolytic enzymes. The hierarchical clustering of proteins in secretome of fungal strains and their co-cultures elucidated differential expressions of lignocellulolytic proteins by the microbial consortium.
Journal of proteomics 08/2012; 75(18):5590-603. · 5.07 Impact Factor
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ABSTRACT: Chromatin is a highly dynamic well organized nucleoprotein complex of DNA and proteins that controls DNA-dependent processes such as transcription, replication, repair and many others. Chromatin structure is regulated by various chromatin associated proteins, post-translational modifications of histones and DNA methylation, but a complete picture of structural changes in chromatin architecture is unclear due to the lack of comprehensive data of chromatin-associated proteins and their bindings to different chromatin regions. This study temporally released chromatin-associated proteins by DNase I and MNase treatment and profiled them by exponentially modified protein abundance index (emPAI) based label-free quantitative proteomics. We identified 694 high confidence proteins, with 160 known chromatin-associated proteins. Identified proteins were functionally classified into histones, non-histones involved in architectural maintenance, proteins involved in DNA replication and repair, transcription machinery, transcription regulation, other chromatin proteins, cell cycle proteins and several novel proteins. Numerous proteins presumed to be chromatin associated were identified and their chromatin interactions were explored. The comprehensive differential chromatin bound proteome might expand our knowledge of the proteins that were associated with different chromatin regions, which could be very useful in elucidating chromatin biology.
Journal of proteomics 07/2012; 75(17):5493-506. · 5.07 Impact Factor
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ABSTRACT: Atrogin-1, a muscle-specific E3 ligase, targets MyoD for degradation through the ubiquitin-proteasome-mediated system. Myostatin, a member of the transforming growth factor-β superfamily, potently inhibits myogenesis by lowering MyoD levels. While atrogin-1 is upregulated by myostatin, it is currently unknown whether atrogin-1 plays a role in mediating myostatin signaling to regulate myogenesis. In this report, we have confirmed that atrogin-1 increasingly interacts with MyoD upon recombinant human myostatin (hMstn) treatment. The absence of atrogin-1, however, led to elevated MyoD levels and permitted the differentiation of atrogin-1(-/-) primary myoblast cultures despite the presence of exogenous myostatin. Furthermore, inactivation of atrogin-1 rescued myoblasts from growth inhibition by hMstn. Therefore, these results highlight the central role of atrogin-1 in regulating myostatin signaling during myogenesis. Currently, there are only two known targets of atrogin-1. Thus, we next characterized the associated proteins of atrogin-1 in control and hMstn-treated C2C12 cell cultures by stably expressing tagged atrogin-1 in myoblasts and myotubes, and sequencing the coimmunoprecipitated proteome. We found that atrogin-1 putatively interacts with sarcomeric proteins, transcriptional factors, metabolic enzymes, components of translation, and spliceosome formation. In addition, we also identified that desmin and vimentin, two components of the intermediate filament in muscle, directly interacted with and were degraded by atrogin-1 in response to hMstn. In summary, the muscle wasting effects of the myostatin-atrogin-1 axis are not only limited to the degradation of MyoD and eukaryotic translation initiation factor 3 subunit f, but also encompass several proteins that are involved in a wide variety of cellular activities in the muscle.
AJP Cell Physiology 06/2012; 303(5):C512-29. · 3.54 Impact Factor
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ABSTRACT: Tianma (Rhizoma gastrodiae) is the dried rhizome of the plant Gastrodia elata Blume (Orchidaceae family). As a medicinal herb in traditional Chinese medicine (TCM) its functions are to control convulsions, pain, headache, dizziness, vertigo, seizure, epilepsy and others. In addition, tianma is frequently used for the treatment of neurodegenerative disorders though the mechanism of action is widely unknown. Accordingly, this study was designed to examine the effects of tianma on the proteome metabolism in differentiated human neuronal SH-SY5Y cells to explore its specific effects on neuronal signaling pathways. Using an iTRAQ (isobaric tags for relative and absolute quantitation)-based proteomics research approach, we identified 2390 modulated proteins, out of which 406 were found to be altered by tianma in differentiated human neuronal SH-SY5Y cells. Based on the observed data, we hypothesize that tianma promotes neuro-regenerative signaling cascades by controlling chaperone/proteasomal degradation pathways (e.g. CALR, FKBP3/4, HSP70/90) and mobilizing neuro-protective genes (such as AIP5) as well as modulating other proteins (RTN1/4, NCAM, PACSIN2, and PDLIM1/5) with various regenerative modalities and capacities related to neuro-synaptic plasticity.
Neurochemistry International 06/2012; 60(8):827-36. · 2.86 Impact Factor
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ABSTRACT: Humoral and tumoral factors collectively promote cancer-induced skeletal muscle wasting by increasing protein degradation. Although several humoral proteins, namely TNFα (tumour necrosis factor α) and IL (interleukin)-6, have been shown to induce skeletal muscle wasting, there is a lack of information regarding the tumoral factors that contribute to the atrophy of muscle during cancer cachexia. Therefore, in the present study, we have characterized the secretome of C26 colon cancer cells to identify the tumoral factors involved in cancer-induced skeletal muscle wasting. In the present study, we show that myostatin, a procachectic TGFβ (transforming growth factor β) superfamily member, is abundantly secreted by C26 cells. Consistent with myostatin signalling during cachexia, treating differentiated C2C12 myotubes with C26 CM (conditioned medium) resulted in myotubular atrophy due to the up-regulation of muscle-specific E3 ligases, atrogin-1 and MuRF1 (muscle RING-finger protein 1), and enhanced activity of the ubiquitin-proteasome pathway. Furthermore, the C26 CM also activated ActRIIB (activin receptor type II B)/Smad and NF-κB (nuclear factor κB) signalling, and reduced the activity of the IGF-I (insulin-like growth factor 1)/PI3K (phosphoinositide 3-kinase)/Akt pathway, three salient molecular features of myostatin action in skeletal muscles. Antagonists to myostatin prevented C26 CM-induced wasting in muscle cell cultures, further confirming that tumoral myostatin may be a key contributor in the pathogenesis of cancer cachexia. Finally, we show that treatment with C26 CM induced the autophagy-lysosome pathway and reduced the number of mitochondria in myotubes. These two previously unreported observations were recapitulated in skeletal muscles collected from C26 tumour-bearing mice.
Biochemical Journal 05/2012; 446(1):23-36. · 4.90 Impact Factor
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ABSTRACT: Solid state fermentation of lignocellulosic biomass by filamentous microorganisms to induced enzyme production has been recognized as an attractive and cost effective technology. The secretion profile of lignocellulolytic enzymes by thermostable filamentous Thermobifida fusca (T. fusca) in solid state fermentation of different lignocellulosic biomasses, such as corn stover, hay; saw dust; sugarcane bagasse; wood chips; and un-dried green plant were explored using label-free exponentially modified protein abundance index (emPAI) based quantitative proteomics. Comparative analyses of T. fusca secretion profiles between cellulose and the various lignocellulosic biomasses showed induced expression of cellulolytic proteins by cellulose, and expression of hemicellulose, pectin and lignin degrading enzymes were induced by lignocellulosic biomasses. The solid state fermentation by T. fusca on lignocellulosic biomasses also revealed increased expressions of various transport proteins and hypothetical proteins. The Bray-Curtis similarity indices, clustering, and multidimensional scaling plot explicated differential protein expressions by T. fusca on different lignocellulosic biomasses, indicating that protein secretion by T. fusca is reliant on substrate complexity.
Journal of proteomics 05/2012; 75(12):3694-706. · 5.07 Impact Factor
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Tiannan Guo,
Lingling Fan,
Wai Har Ng,
Yi Zhu,
Mengfatt Ho,
Wei Keat Wan,
Kiat Hon Lim,
Whee Sze Ong,
Sze Sing Lee,
Shiang Huang,
Oi Lian Kon, Siu Kwan Sze
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ABSTRACT: Gastric cancer remains highly fatal due to a dearth of diagnostic biomarkers for early stage disease and molecular targets for therapy. Plasma membrane proteins, including cluster of differentiation (CD) proteins and receptor tyrosine kinases (RTKs), are a rich reservoir of biomarkers. Recognizing that interrogating plasma membrane proteins individually overlooks extensive interactions among them, we have systematically investigated the membrane proteomes and transcriptomes of six gastric cancer cell lines. Our data revealed aberrantly high expression of proteins whose functions accurately reflect the clinical phenotype of gastric cancer, and prioritized critical RTKs and CD proteins in gastric cancer. Expression of selected surface proteins was confirmed by flow cytometry and immunostaining of clinical gastric cancer tissues. Close to 90% of the gastric cancer tissues in a cohort showed up-regulation of at least one of four proteins, that is, MET, EPHA2, FGFR2, and CD104/ITGB4. All intestinal type gastric cancer tumors in this cohort overexpressed at least one of a panel of three proteins, MET, FGFR2, and EPHA2. This study reports the first quantitative global landscape of the surface proteome of gastric cancer cells and provides a shortlist of gastric cancer biomarkers.
Journal of Proteome Research 04/2012; · 5.11 Impact Factor
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ABSTRACT: Despite decades of intensive research, there is still no effective treatment for ischemia/reperfusion (I/R) injury, an important corollary in the treatment of ischemic disease. I/R injury is initiated when the altered biochemistry of cells after ischemia is no longer compatible with oxygenated microenvironment (or reperfusion). To better understand the molecular basis of this alteration and subsequent incompatibility, we assessed the temporal and quantitative alterations in the cardiac proteome of a mouse cardiac I/R model by an iTRAQ approach at 30 min of ischemia, and at 60 or 120 min reperfusion after the ischemia using sham-operated mouse heart as the baseline control. Of the 509 quantified proteins identified, 121 proteins exhibited significant changes (p-value<0.05) over time and were mostly clustered in eight functional groups: Fatty acid oxidation, Glycolysis, TCA cycle, ETC (electron transport chain), Redox Homeostasis, Glutathione S-transferase, Apoptosis related, and Heat Shock proteins. The first four groups are intimately involved in ATP production and the last four groups are known to be important in cellular antioxidant activity. During ischemia and reperfusion, the short supply of oxygen precipitates a pivotal metabolic switch from aerobic metabolism involving fatty acid oxidation, TCA, and phosphorylation to anaerobic metabolism for ATP production and this, in turn, increases reactive oxygen species (ROS) formation. Therefore the implication of these 8 functional groups suggested that ischemia-reperfusion injury is underpinned in part by proteomic alterations. Reversion of these alterations to preischemia levels took at least 60 min, suggesting a refractory period in which the ischemic cells cannot adjust to the presence of oxygen. Therefore, therapeutics that could compensate for these proteomic alterations during this interim refractory period could alleviate ischemia-reperfusion injury to enhance cellular recovery from an ischemic to a normoxic microenvironment. Among the perturbed proteins, Park7 and Ppia were selected for further investigation of their functions under hypoxia. The results show that Park7 plays a key role in regulating antioxidative stress and cell survival, and Ppia may function in coping with the unfolded protein stress in the I/R condition.
Journal of Proteome Research 03/2012; 11(4):2331-46. · 5.11 Impact Factor
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ABSTRACT: Gastrodia elata (tianma) is a traditional Chinese herbal medicine (TCM) often used for the treatment of cerebrovascular diseases. In this study, we investigated the effects of tianma on the brain protein metabolism by quantitative proteomics to gain evidence for a direct relationship between tianma treatment and brain functions. One-year-old rats were treated with tianma (~2.5 g/kg/day) for 3months and the brain tissue proteome was analyzed by using the iTRAQ (isobaric tag for relative and absolute quantification) technology. According to our results, the long-term treatment with tianma could modulate the brain protein metabolism at the proteome level by down-regulating the expressions of various proteins, such as Gnao1 and Dctn2, which are related to neuronal growth cone control and synaptic activities. In addition, tianma treatment also induced the up-regulation of molecular chaperons and proteins related to the misfolded protein response, like Anxa5, and also other proteins involved in Huntington's disease (HD) (e.g. Pacsin1 and Arf3). Concluding, tianma could eventually contribute to activities related to synaptic plasticity and neuro-restorative processes and thus might be a novel candidate agent for the treatment of neurodegenerative diseases by regulating the brain proteome.
Journal of proteomics 03/2012; 75(8):2468-79. · 5.07 Impact Factor
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ABSTRACT: Deamidation of asparaginyl residues in proteins produces a mixture of asparaginyl, n-aspartyl, and isoaspartyl residues, which affects the proteins' structure, function, and stability. Thus, it is important to identify and quantify the products to evaluate the effects in biological systems. It is still a challenging task to distinguish between the n-Asp and isoAsp deamidation products in a proteome-wide analysis because of their similar physicochemical properties. The quantification of the isomeric deamidated peptides is also rather difficult because of their coelution/poor separation in reverse-phase liquid chromatography (RPLC). We here propose a RP-ERLIC-MS/MS approach for separating and quantifying on a proteome-wide scale the three products related to deamidation of the same peptide. The key to the method is the use of RPLC in the first dimensional separation and ERLIC (electrostatic repulsion-hydrophilic interaction chromatography) in the second, with direct online coupling to tandem MS. The coelution of the three deamidation-related peptides in RPLC is then an asset, as they are collected in the same fraction. They are then separated and identified in the second dimension with ERLIC, which separates peptides on the basis of both pI and GRAVY values. The coelution of the three products in RPLC and their efficient separation in ERLIC were validated using synthetic peptides, and the performance of ERLIC-MS/MS was tested using peptide mixtures from two proteins. Applying this sequence to rat liver tissue, we identified 302 unique N-deamidated peptides, of which 20 were identified via all three deamidation-related products and 70 of which were identified via two of them.
Journal of Proteome Research 03/2012; 11(3):1804-11. · 5.11 Impact Factor
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ABSTRACT: Trichoderma reesei is a mesophilic, filamentous fungus, and it is a major industrial source of cellulases, but its lignocellulolytic protein expressions on lignocellulosic biomass are poorly explored at present. The extracellular proteins secreted by T. reesei QM6a wild-type and hypercellulolytic mutant Rut C30 grown on natural lignocellulosic biomasses were explored using a quantitative proteomic approach with 8-plex high throughput isobaric tags for relative and absolute quantification (iTRAQ) and analyzed by liquid chromatography tandem mass spectrometry. We quantified 230 extracellular proteins, including cellulases, hemicellulases, lignin-degrading enzymes, proteases, protein-translocating transporter, and hypothetical proteins. Quantitative iTRAQ results suggested that the expressions and regulations of these lignocellulolytic proteins in the secretome of T. reesei wild-type and mutant Rut C30 were dependent on both nature and complexity of different lignocellulosic carbon sources. Therefore, we discuss here the essential lignocellulolytic proteins for designing an enzyme mixture for optimal lignocellulosic biomass hydrolysis.
Molecular & Cellular Proteomics 02/2012; 11(7):M111.012419. · 7.40 Impact Factor
