Differential Protein Profiles Research Articles

Proteomic Identification and Analysis of Human Endometrial Proteins Associated with Unexplained Infertility

Unexplained infertility (UI) represents about 25-30% of all known types of female infertility. To date, UI remains an unsolved problem in a subgroup of infertile but otherwise healthy women and the molecular causes of UI are not yet known. In the current study, we have compared and analyzed the proteomic profile of receptive phase (LH+7) endometrium from fertile and infertile women, with a view to identify the appropriate protein target signatures that may be responsible for defective endometrial receptivity as a cause of UI. 12 Differentially expressed proteins (8 up-regulated and 4 down-regulated) were identified by Liquid Chromatography-Mass Spectrometric analysis. These differentially expressed proteins were involved in immunological response, glycolytic pathway, lipid metabolism, blood agglutination, protein synthesis, molecular chaperone, antioxidant system, mitochondrial ATP generation, and Ca+2 signalling. The expression of four differentially expressed proteins such as HSPβ-1, Apolipoprotein-A1, IGK@ protein, and RPLP2 were further validated by immunoblotting and immuno-histochemical analysis in separate biopsy samples, and also in in-vitro experimental model of decidualization of human endometrial stromal cells (hESCs). This study explored several proteins that may have functional significance as regards the initiation and maintenance of the window of receptivity. Results of this study might be helpful in understanding the molecular basis of endometrial defects in a subset of infertile women having normal ovulation and hormonal profile. To our understanding, this is the first study to demonstrate the differential endometrial protein profiling in women with endometrium basedunexplained infertility.

Diet-Induced Over-Expression of Flightless-I Protein and Its Relation to Flightlessness in Mediterranean Fruit Fly, Ceratitis capitata

The Mediterranean fruit fly (medfly), Ceratitis capitata is among the most economically important pests worldwide. Understanding nutritional requirement helps rearing healthy medfly for biocontrol of its population in fields. Flight ability is a high priority criterion. Two groups of medfly larvae were reared with two identical component diets except one with fatty acids (diet A) and another without it (diet B). Adults from larvae reared on diet B demonstrated 20±8% of normal flight ability, whereas those from larvae reared on diet A displayed full flight ability of 97±1%. Proteomes were profiled to compare two groups of medfly pupae using shotgun proteomics to study dietary effects on flight ability. When proteins detected in pupae A were compared with those in pupae B, 233 and 239 proteins were, respectively, under- and over-expressed in pupae B, while 167 proteins were overlapped in both pupae A and B. Differential protein profiles indicate that nutritional deficiency induced over-expression of flightless-I protein (fli-I) in medfly. All proteins were subjected to Ingenuity Pathway Analysis (IPA) to create 13 biological networks and 17 pathways of interacting protein clusters in human ortholog. Fli-I, leucine-rich repeat (LRR)-containing G protein-coupled receptor 2, LRR protein soc-2 and protein wings apart-like were over-expressed in pupae B. Inositol-1,4,5-trisphosphate receptor, protocadherin-like wing polarity protein stan and several Wnt pathway proteins were under-expressed in pupae B. These results suggest down-regulation of the Wnt/wingless signaling pathway, which consequently may result in flightlessness in pupae B. The fli-I gene is known to be located within the Smith-Magenis syndrome (SMS) region on chromosome 17, and thus, we speculate that nutritional deficiency might induce over-expression of fli-I (or fli-I gene) and be associated with human SMS. However, more evidence would be needed to confirm our speculation.

Selectivity analysis of single binder assays used in plasma protein profiling

The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from antibody-coupled beads by analysis of their eluates by Western blots and MS. We demonstrate selective antibody binding in complex samples with antibodies toward a set of chosen proteins with different abundance in plasma and serum, and illustrate the need to adjust sample and bead concentrations accordingly. The presented approach will serve as an important tool for resolving differential protein profiles from antibody arrays within plasma biomarker discoveries.

Proteomic Differences between Tellurite-Sensitive and Tellurite–Resistant E.coli

Tellurite containing compounds are in use for industrial processes and increasing delivery into the environment generates specific pollution that may well result in contamination and subsequent potential adverse effects on public health. It was the aim of the current study to reveal mechanism of toxicity in tellurite-sensitive and tellurite-resistant E. coli at the protein level.In this work an approach using gel-based mass spectrometrical analysis to identify a differential protein profile related to tellurite toxicity was used and the mechanism of ter operon-mediated tellurite resistance was addressed. E. coli BL21 was genetically manipulated for tellurite-resistance by the introduction of the resistance-conferring ter genes on the pLK18 plasmid. Potassium tellurite was added to cultures in order to obtain a final 3.9 micromolar concentration. Proteins from tellurite-sensitive and tellurite-resistant E. coli were run on 2-D gel electrophoresis, spots of interest were picked, in-gel digested and subsequently analysed by nano-LC-MS/MS (ion trap). In addition, Western blotting and measurement of enzymatic activity were performed to verify the expression of certain candidate proteins.Following exposure to tellurite, in contrast to tellurite-resistant bacteria, sensitive cells exhibited increased levels of antioxidant enzymes superoxide dismutases, catalase and oxidoreductase YqhD. Cysteine desulfurase, known to be related to tellurite toxicity as well as proteins involved in protein folding: GroEL, DnaK and EF-Tu were upregulated in sensitive cells. In resistant bacteria, several isoforms of four essential Ter proteins were observed and following tellurite treatment the abovementioned protein levels did not show any significant proteome changes as compared to the sensitive control.The absence of general defense mechanisms against tellurite toxicity in resistant bacteria thus provides further evidence that the four proteins of the ter operon function by a specific mode of action in the mechanism of tellurite resistance probably involving protein cascades from antioxidant and protein folding pathways.

Comparative proteomic analysis of thiol proteins in the liver after oxidative stress induced by diethylnitrosamine

Conversion of protein –SH groups to disulfides is an early event during protein oxidation, which has prompted great interest in the study of thiol proteins. Chemical carcinogenesis is strongly associated with the formation of reactive oxygen species (ROS). The goal of this study was to detect thiol proteins that are sensitive to ROS generated during diethylnitrosamine (DEN) metabolism in the rat liver. DEN has been widely used to induce experimental hepatocellular carcinoma. We used modified redox-differential gel electrophoresis (redox-DIGE method) and mass spectrometry MALDI-TOF/TOF to identify differential oxidation protein profiles associated with carcinogen exposure. Our analysis revealed a time-dependent increase in the number of oxidized thiol proteins after carcinogen treatment; some of these proteins have antioxidant activity, including thioredoxin, peroxirredoxin 2, peroxiredoxin 6 and glutathione S-transferase alpha-3. According to functional classifications, the identified proteins in our study included chaperones, oxidoreductases, activity isomerases, hydrolases and other protein-binding partners. This study demonstrates that oxidative stress generated by DEN tends to increase gradually through DEN metabolism, causes time-dependent necrosis in the liver and has an oxidative effect on thiol proteins, thereby increasing the number of oxidized thiol proteins. Furthermore, these events occurred during the hepatocarcinogenesis initiation period.

Comparative differential proteomic profiles of nonfailing and failing hearts after in vivo thoracic aortic constriction in mice overexpressing FKBP12.6

Chronic pressure overload (PO) induces pathological left ventricular hypertrophy (LVH) leading to congestive heart failure (HF). Overexpression of FKBP12.6 (FK506-binding protein [K]) in mice should prevent Ca2+-leak during diastole and may improve overall cardiac function. In order to decipher molecular mechanisms involved in thoracic aortic constriction (TAC)-induced cardiac remodeling and the influence of gender and genotype, we performed a proteomic analysis using two-dimensional differential in-gel electrophoresis (2D-DIGE), mass spectrometry, and bioinformatics techniques to identify alterations in characteristic biological networks. Wild-type (W) and K mice of both genders underwent TAC. Thirty days post-TAC, the altered cardiac remodeling was accompanied with systolic and diastolic dysfunction in all experimental groups. A gender difference in inflammatory protein expression (fibrinogen, α-1-antitrypsin isoforms) and in calreticulin occurred (males > females). Detoxification enzymes and cytoskeletal proteins were noticeably increased in K mice. Both non- and congestive failing mouse heart exhibited down- and upregulation of proteins related to mitochondrial function and purine metabolism, respectively. HF was characterized by a decrease in enzymes related to iron homeostasis, and altered mitochondrial protein expression related to fatty acid metabolism, glycolysis, and redox balance. Moreover, two distinct differential protein profiles characterized TAC-induced pathological LVH and congestive HF in all TAC mice. FKBP12.6 overexpression did not influence TAC-induced deleterious effects. Huntingtin was revealed as a potential mediator for HF. A broad dysregulation of signaling proteins associated with congestive HF suggested that different sets of proteins could be selected as useful biomarkers for HF progression and might predict outcome in PO-induced pathological LVH.

Abstract 1080: Nanotechnology-enhanced proteomics analysis of a unique prospective collection of blood samples for the discovery and validation of early detection biomarkers for Non-Small Cell Lung Cancer (NSCLC).

Abstract BACKGROUND. Early identification of individuals at high risk to develop lung cancer would be a crucial requirement for a successful prevention strategy. The novelty of this study lies in our ability to discover and validate biomarkers for subclinical disease in blood samples obtained from healthy individuals up to 25 years prior to lung cancer diagnosis. Plasma samples were collected from healthy individuals who later developed Non-Small Cell Lung Cancer (NSCLC) (cases) matched with individuals who did not develop cancer after the same period of follow up (controls). We studied the influence of smoking status (non-smokers, smokers and former smokers) on the pattern of blood borne biomarkers in order to test the hypothesis that the developing tumor might have distinct molecular origins in the three classes. METHODS. Study subjects were drawn from the longitudinal Physicians’ Health Study (PHS). We analyzed plasma samples from 42 cases and 42 controls (16 to 284 month follow up). Small proteins and peptides were purified from plasma using N-isopropylacrylamide (NIPAm) core shell hydrogel particles functionalized with Cibacron blue F3GA and vinylsulfonic acid as chemical baits. Proteins eluted from the particles were analyzed using LTQ-Orbitrap mass spectrometer (Thermo Fisher). RESULTS. 67, 83 and 75 candidate biomarkers of early NSCLC were identified for non-smokers, former smokers and smokers, respectively. An optimal combination of predictive biomarkers was selected for each group. Receiver-operating characteristic (ROC) curves were utilized to evaluate the predictive value of selected biomarkers. In non-smokers, a panel of 8 proteins including Keratin 6B and endostatin had 82% sensitivity and 91% specificity (AUC 0.926) in predicting an individual developing cancer. In former smokers, a combination of 8 proteins (including malic enzyme 2 and mitochondrial malate dehydrogenase) had 88% sensitivity and 82% specificity (AUC 0.901). In smokers, 10 selected biomarkers (including fructose-bisphosphate aldolase A and glyceraldehyde-3-phosphate dehydrogenase) had 75% sensitivity and 75% specificity (AUC 0.833). Differential plasma protein profiles of smokers or former smokers that developed cancer included a high number of proteins involved in the response to high oxidative stress. Oxidative stress, together with inflammation, could be the driving pre requisite for lung cancer development in individuals exposed to smoke. Interestingly, endostatin (a known inhibitor of angiogenesis) was over-represented (Chi-Square p < 0.031) in cases versus controls independent of smoking status. The predictive value of identified candidate biomarkers will be verified in a larger cohort of independent blinded samples from the same prospective collection study. Citation Format: Claudia Fredolini, Alessandra Luchini, Luisa Paris, Ruben Magni, Emanuel F. Petricoin, Salima Darakjy, J. Michael Gaziano, Howard D. Sesso, Lance A. Liotta, Frederica P. Perera. Nanotechnology-enhanced proteomics analysis of a unique prospective collection of blood samples for the discovery and validation of early detection biomarkers for Non-Small Cell Lung Cancer (NSCLC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1080. doi:10.1158/1538-7445.AM2013-1080

Acute administration of ethanol reduces apoptosis following ischemic stroke in rats

In recent studies, acute ethanol administration appears to play a neuroprotective role during ischemic stroke. We sought to confirm these findings by identifying if ethanol-derived neuroprotection is associated with a reduction in apoptosis.Ethanol at 0.5 and 1.5g/kg doses was given by intraperitoneal injections to Sprague-Dawley rats after 2h of middle cerebral artery (MCA) occlusion, followed by reperfusion. We quantified apoptotic cell death in each of the treatment groups with ELISA, and measured pro- and anti-apoptotic protein expression with Western blot analysis.Cell death was significantly increased in rats after ischemia and was subsequently significantly reduced by the administration of 1.5g/kg of ethanol. We found that the 1.5g/kg dose promoted the expression of pro-survival factors and decreased the expression of apoptotic proteins at 3h after reperfusion. This effect was maintained at 24h for Caspase-3 and apoptosis-inducing factor (AIF), although not for Bcl-2, Bcl-xL, and Bcl-2-associated X (Bax). Administration of 0.5g/kg of ethanol was not as effective in regulating protein expression as the 1.5g/kg dose.Our study suggests that administration of ethanol at a dose of 1.5g/kg after stroke – which provides rat blood alcohol levels equivalent to the legal driving limit – produces a differential protein profile, with increased expression of anti-apoptotic proteins and decrease in pro-apoptotic factors. This results in a significant reduction of neuronal apoptosis and is neuroprotective in ischemia-reperfusion injury.

Abstract WP159: Acute Administration of Ethanol Reduced Brain Apoptosis by Regulating Expression of Apoptotic Proteins in Ischemic Stroke

Objectives: In our recent studies, acute ethanol administration appears to play a neuroprotective role after the onset of ischemic stroke by reducing infarct volume and behavioral dysfunction ( Stroke. 2012 43(1):205-10 ). In the present study, we sought to identify whether the ethanol-derived neuroprotection is associated with a reduction in apoptosis. Methods: Ethanol was given by intraperitoneal injections to Sprague-Dawley rats after 2 hours of middle cerebral artery (MCA) occlusion, followed by reperfusion. The actual degree of apoptotic cell death in control, ischemic rats, and ischemic rats treated with 1.5 g/kg ethanol was determined by the Cell Death Detection ELISA Assay (Roche). Levels of pro-apoptotic (Caspase-3, Bcl-2-associated X “BAX”, and Apoptosis-Inducing Factor “AIF”) and anti-apoptotic proteins (Bcl-2 and Bcl-xL) were determined by Western blot at 3 and 24 hours after reperfusion. Results: Rats treated with ethanol at the 1.5 g/kg dosage demonstrated significantly reduced cell death as compared to those of stroke without treatment. Acute ethanol administration at the 1.5 g/kg dose significantly promoted the expression of anti-apoptotic factors and decreased the expression of pro-apoptotic proteins at 3 hours after reperfusion. This effect was maintained at 24 hours for Caspase-3 and AIF, but not for Bcl-2, Bcl-xL, and Bax. We further demonstrated that administration of low dose of ethanol at 0.5 g/kg was not as effective in regulating protein expression as the 1.5 g/kg dose (1.5 g/kg dose being equivalent to the legal driving limit). Conclusions: Our study suggests that administration of ethanol at a 1.5 g/kg dosage after onset of stroke reduces apoptosis by creating a differential protein profile, with increased expression of anti-apoptotic proteins and decreased expression of pro-apoptotic ones. This study helps in validating ethanol as a new therapeutic approach in neuroprotection after stroke, and proposes a possible mechanism for its anti-apoptotic effect on neuronal cells.

Plasma biomarkers for distinguishing etiologic subtypes of thoracic aortic aneurysm disease

Thoracic aortic aneurysms (TAAs) develop through an asymptomatic process resulting in gross dilation that progresses to rupture if left undetected and untreated. If detected, patients with TAA are followed over time until the risk of rupture outweighs the risk of surgical repair. Current methodologies for tracking TAA size are limited to expensive computed tomography or magnetic resonance imaging because no acceptable population screening tools are currently available. Previous studies from this laboratory and others have identified differential protein profiles for the matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs), in ascending TAA tissue from patients with bicuspid aortic valves (BAVs), versus patients with idiopathic degenerative disease and a tricuspid aortic valve (TAV). In addition, altered microRNA (miR) expression levels have also been reported in TAAs compared with normal aortic tissue. The objective of our study was to identify circulating factors within plasma that could serve as potential biomarkers for distinguishing etiologic subtypes of aneurysm disease. Ascending TAA tissue and plasma specimens were obtained from patients with BAV (n=21) and TAV (n=21) at the time of surgical resection. The protein abundance of key MMPs (1, 2, 3, 8, and 9), TIMPs (1, 2, 3, and 4), and miRs (1, 21, 29a, 133a, 143, and 145) was examined using a multianalyte protein profiling system or by quantitative polymerase chain reaction, respectively. Results were compared with normal aortic tissue and plasma obtained from patients without aortic disease (n=10). Significant (P<.05) differences in standardized miR-1 and miR-21 abundance between BAV and TAV aortic tissue samples and different tissue and plasma profiles of analyte differences from normal aorta where observed between the BAV and TAV groups. Linear regression analysis revealed significant linear relationships in plasma and tissue measurements only for MMP-8 and TIMP-1, TIMP-3, and TIMP-4 (P<.05). Receiver operator curve analysis revealed specific cassettes of analytes predictive of TAA disease. Relative to normal aorta, BAV proteolytic balance was significantly increased for MMP-1, MMP-2, and MMP-7, and for decreased MMP-8 and MMP-9. In contrast, TAV proteolytic balance relative to normal aorta was significantly increased only for MMP-1 and decreased for MMP-8 and MMP-9. Taken together, these unique data demonstrate differential plasma profiles of MMPs, TIMPs, and miRs in ascending TAA specimens from patients with BAV and TAV. These results suggest that circulating biomarkers may form the foundation for a broader platform of biomarkers capable of detecting the presence of TAA using a simple blood test and may also be useful in personalized strategies to distinguish between etiologic subtypes of TAAs in patients with aneurysm disease.

An iTRAQ approach to quantitative proteome analysis of cerebrospinal fluid from patients with tuberculous meningitis

To study the cerebrospinal fluid (CSF) protein profiles of tuberculous meningitis (TBM) and discover potential biomarkers for TBM, differential expression of proteins in the CSF of patients with TBM, patients with cryptococcal meningitis, and a control group were compared using isobaric tags for relative and absolute quantitation labelling (iTRAQ) coupled with 2-dimensional liquid chromatography-tandem mass spectrometry (LC-MS). As a result, a total of 208 unique proteins with a molecular weight ranging from 10 KD to 135 KD were identified and quantified in CSF samples from patients with TBM. Of the proteins, 9 were expressed at levels differing 2.0 fold, 6 were up-regulated, and 3 were down-regulated. These proteins appear to be involved in calcium ion binding, lipoprotein metabolism, immune response, and signal conduction. Two differentially expressed proteins were identified using ELISA. The present study represents the successful use of iTRAQ to examine CSF from patients with TBM. The differentially expressed proteins identified may be potential diagnostic biomarkers and provide valuable insight into the underlying mechanisms of TBM. This study also demonstrated that the differential protein profiles of diseases can be quickly determined using iTRAQ-LC-MS, a potential method for quantitative comparative proteomics.

Serum proteins in chronic hepatitis B patients treated with peginterferon alfa-2b

To study the differential protein profile in serum of hepatitis B patients. Serum samples were obtained from patients with chronic hepatitis B who were receiving peginterferon alfa-2b. The serum samples were subjected to albumin depletion and analyzed by two-dimensional gel electrophoresis (2-DE). Differentially expressed protein spots were identified by electrospray ionization-quadrupole time-of-flight mass spectrometry. Alpha-2-HS-glycoprotein, complement component C3c and CD5 antigen were further analyzed by an enzyme-linked immunosorbent assay and immunonephelometry. Nineteen patients with HBeAg-positive chronic hepatitis B (CHB) were studied. These patients were followed for at least 1 year after treatment and were classified according to their treatment response: responders (n = 9) and non-responders (n = 10). 2-DE and MS/MS analysis were performed to compare the serum proteins before initiating peginterferon alfa-2b. From the quantitative analysis of the 2-D gel, 7 proteins were detected between the two groups at different levels before treatment. Among these potential candidates, serum levels of alpha-2-HS-glycoprotein, complement component C3c and CD5 antigen-like precursor were further analyzed. In the validation phase, 23 subjects, 9 sustained responders and 14 non-responders, were recruited. Interestingly, the levels of alpha-2-HS-glycoprotein and complement component C3c were elevated in the serum of the non-responders compared to the responders. Serum alpha-2-HS-glycoprotein and complement component C3c may be potential serum biomarkers in predicting the treatment response of peginterferon alfa-2b in patients with CHB prior to treatment.

Differential protein profiling of renal cell carcinoma urinary exosomes

Renal cell carcinoma (RCC) accounts for about 3% of all human malignancies and its incidence is increasing. There are no standard biomarkers currently used in the clinical management of patients with renal cell carcinoma. A promising strategy for new biomarker detection is comparative proteomics of urinary exosomes (UE), nanovesicles released by every epithelial cell facing the urinary space, enriched in renal proteins and excluding high-abundance plasmatic proteins, such as albumin. Aim of the work is to establish the protein profile of exosomes isolated from urines of RCC patient compared with control subjects. We enrolled 29 clear cell RCC patients and 23 control healthy subjects (CTRL), age and sex-matched, for urine collection and vesicle isolation by differential centrifugation. Such vesicles were morphologically and biochemically characterized and proved to share exosome properties. Proteomic analysis, performed on 9 urinary exosome (UE) pooled samples by gel based digestion followed by LC-MS/MS, led to the identification of 261 proteins from CTRL subject UE and 186 from RCC patient UE, and demonstrated that most of the identified proteins are membrane associated or cytoplasmic. Moreover, about a half of identified proteins are not shared between RCC and control UE. Starting from these observations, and from the literature, we selected a panel of 10 proteins, whose UE differential content was subjected to immunoblotting validation. Results show for the first time that RCC UE protein content is substantially and reproducibly different from control UE, and that these differences may provide clues for new RCC biomarker discovery.

Proteomic and metabolomic responses to connexin43 silencing in primary hepatocyte cultures

Freshly established cultures of primary hepatocytes progressively adopt a foetal-like phenotype and display increased production of connexin43. The latter is a multifaceted cellular entity with variable subcellular locations, including the mitochondrial compartment. Cx43 forms hemichannels and gap junctions that are involved in a plethora of physiological and pathological processes, such as apoptosis. The present study was conducted with the goal of shedding more light onto the role of connexin43 in primary hepatocyte cultures. Connexin43 expression was suppressed by means of RNA interference technology, and the overall outcome of this treatment on the hepatocellular proteome and metabolome was investigated using tandem mass tag-based differential protein profiling and (1)H NMR spectroscopy, respectively. Global protein profiling revealed a number of targets of the connexin43 knock-down procedure, including mitochondrial proteins (heat shock protein 60, glucose-regulated protein 75, thiosulphate sulphurtransferase and adenosine triphosphate synthase) and detoxifying enzymes (glutathione S-transferase μ 2 and cytochrome P450 2C70). At the metabolomic level, connexin43 silencing caused no overt changes, though there was some evidence for a subtle increase in intracellular glycine quantities. Collectively, these data could further substantiate the established existence of a mitochondrial connexin pool and could be reconciled with the previously reported involvement of connexin43 signalling in spontaneously occurring apoptosis in primary hepatocyte cultures.

P135 Defining the intracellular innate immune synapse

Introduction Innate immunity to RNA virus infection is triggered when the cytosolic pathogen recognition receptor RIG-I engages viral RNA in infected cells. RIG-I pathway signaling is transmitted by the RIG-I adaptor protein MAVS, which resides on mitochondria, peroxisomes, and the mitochondrial-associated membrane (MAM), a distinct membrane that links ER to mitochondria. During RNA virus infection, RIG-I is recruited into the MAM where it binds MAVS and drives the actions of a signalosome that mediates downstream induction of antiviral, proinflammatory, and immunomodulatory genes that impart control of infection and immunity. MAM-tethering to mitochondria and peroxisomes coordinates MAVS localization to form a signaling synapse between membranes. The importance of the MAM within this “innate immune synapse” is highlighted by the fact that the hepatitis C virus (HCV) NS3/4A protease cleaves MAVS on the MAM, but not the mitochondria, to evade immunity. Methods To identify the components that regulate formation and function of the innate immune synapse and the MAVS signalosome, we characterized the proteome of MAM, ER, and cytosol subcellular fractions from uninfected cells and from cells with either chronic (HCV) or acute (Sendai) RNA virus infections. Results Comparative analysis of protein trafficking dynamics during both chronic and acute infection reveals differential protein profiles in the MAM compartment under RIG-I pathway activation. We also identified molecules recruited to the MAM in both chronic and acute RNA viral infections representing proteins that drive immunity and/or regulate viral replication. Conclusion Our proteomic analysis reveals dynamic cross-talk between subcellular compartments during both acute and chronic RNA virus infection, and demonstrates the importance of the MAM as a central platform that coordinates innate immune signaling to initiate immunity against RNA virus infection.

Preliminary analysis of differentially expressed proteins of clear-cell renal cell carcinoma by comparative proteomic technologies

To explore the different expression of proteins between human clear-cell renal cell carcinoma (ccRCC) cell line RLC-310 and normal renal proximal tubule epithelial cell line HK-2, and to search new differentially expressed proteins of RCC. RLC-310 and HK-2 cells were cultured in vitro. The total proteins were separated by ProteomeLab PF 2D protein fractionation system and the differential expression protein fractions of the two cell lines were analyzed and identified by capillary liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS). RT-PCR and Western blot were used to confirm the representative differential expression at mRNA and protein levels. One hundred and ninty-six differentially expressed proteins were identified. These differentially expressed proteins involved in many aspects, including cell proliferation and anti-apoptosis, energy metabolism, mitochondria reduction and oxidation, oxidative stress and resistance, cell signaling, invasion and adhesion, cytoskeleton and motion, neovascularization, etc. Except for previously reported RCC associated proteins: annexin A2, fatty acid-binding protein, vimentin, fibronectin, and so on, Septin-9 was firstly found highly expressed in RLC-310 cells when compared with that in the HK-2 cells. Moreover, the overexpression of Septin-9 was confirmed by RT-PCR and Western blot analysis at both mRNA and protein levels (P < 0.05). The human ccRCC cell line RLC-310 cells display differential protein profiles compared with those of the normal renal cell line HK-2 cells. The identified differential expression proteins are involved in many aspects of RCC development. It is worth further study and elucidate the molecular mechanisms of RCC. The representative differential protein Septin-9 deserves further study its role in the angiogenesis of ccRCC.

Spermatogenesis in mammals: proteomic insights

Spermatogenesis is a highly sophisticated process involved in the transmission of genetic heritage. It includes halving ploidy, repackaging of the chromatin for transport, and the equipment of developing spermatids and eventually spermatozoa with the advanced apparatus (e.g., tightly packed mitochondrial sheat in the mid piece, elongating of the tail, reduction of cytoplasmic volume) to elicit motility once they reach the epididymis. Mammalian spermatogenesis is divided into three phases. In the first the primitive germ cells or spermatogonia undergo a series of mitotic divisions. In the second the spermatocytes undergo two consecutive divisions in meiosis to produce haploid spermatids. In the third the spermatids differentiate into spermatozoa in a process called spermiogenesis. Paracrine, autocrine, juxtacrine, and endocrine pathways all contribute to the regulation of the process. The array of structural elements and chemical factors modulating somatic and germ cell activity is such that the network linking the various cellular activities during spermatogenesis is unimaginably complex. Over the past two decades, advances in genomics have greatly improved our knowledge of spermatogenesis, by identifying numerous genes essential for the development of functional male gametes. Large-scale analyses of testicular function have deepened our insight into normal and pathological spermatogenesis. Progress in genome sequencing and microarray technology have been exploited for genome-wide expression studies, leading to the identification of hundreds of genes differentially expressed within the testis. However, although proteomics has now come of age, the proteomics-based investigation of spermatogenesis remains in its infancy. Here, we review the state-of-the-art of large-scale proteomic analyses of spermatogenesis, from germ cell development during sex determination to spermatogenesis in the adult. Indeed, a few laboratories have undertaken differential protein profiling expression studies and/or systematic analyses of testicular proteomes in entire organs or isolated cells from various species. We consider the pros and cons of proteomics for studying the testicular germ cell gene expression program. Finally, we address the use of protein datasets, through integrative genomics (i.e., combining genomics, transcriptomics, and proteomics), bioinformatics, and modelling.

Differential protein profile in zebrafish (Danio rerio) brain under the joint exposure of methyl parathion and cadmium

As different chemicals, methyl parathion (MP) and cadmium (Cd) can induce neurotoxicity on the brain of aquatic ecosystems. This study aims to explore the differential expression proteins in the brain induced by their joint stress and their joint effects, which are poorly reported, and devotes finding novel biomarkers for monitoring their contamination in water and assessing their neurological effects. The bioaccumulation of MP and Cd in tissues after 96h of exposure was first analyzed by GC and inductively coupled plasma-MS to provide insights into the interaction. Protein profile changes in the brains of the zebrafish (Danio rerio) exposed to MP and Cd were further investigated using the proteomic approach. The correlation of gene expression on the transcription level of mRNA and the translation level of protein was examined by real-time quantitative PCR and Western blotting analysis. It showed that Cd and MP have an interaction on their bioaccumulation, which suggests that their joint effect over 96h might be antagonistic. Proteomics revealed that 22 protein spots changed their expression levels under stress, of which 16 proteins were identified using MS. These proteins were involved in oxidation/reduction, metabolism, energy production, receptor activity, and cytoskeleton assembly. Among them, five proteins with a remarkable abundance change are significantly suggested to play important roles in the joint effect. This work demonstrates that there exists an interaction between MP and Cd toxicities, which may aid in our understanding of the mechanism of neurotoxicity induced by joint stress. The results may also provide the possibility of the establishment of candidate biomarkers for monitoring MP and Cd contamination in water.

Candidate Hippocampal Biomarkers of Susceptibility and Resilience to Stress in a Rat Model of Depression

Susceptibility to stress plays a crucial role in the development of psychiatric disorders such as unipolar depression and post-traumatic stress disorder. In the present study the chronic mild stress rat model of depression was used to reveal stress-susceptible and stress-resilient rats. Large-scale proteomics was used to map hippocampal protein alterations in different stress states. Membrane proteins were successfully captured by two-phase separation and peptide based proteomics. Using iTRAQ labeling coupled with mass spectrometry, more than 2000 proteins were quantified and 73 proteins were found to be differentially expressed. Stress susceptibility was associated with increased expression of a sodium-channel protein (SCN9A) currently investigated as a potential antidepressant target. Differential protein profiling also indicated stress susceptibility to be associated with deficits in synaptic vesicle release involving SNCA, SYN-1, and AP-3. Our results indicate that increased oxidative phosphorylation (COX5A, NDUFB7, NDUFS8, COX5B, and UQCRB) within the hippocampal CA regions is part of a stress-protection mechanism.

Proteomic Analysis of Developing Somatic Embryos of Coffea arabica

Differential protein profiles of three stages of somatic embryogenesis, including globular, torpedo, and cotyledonary somatic embryos, of Coffea arabica cv. Catuaí Vermelho were analyzed in an attempt to better understand somatic embryogenesis in coffee plants. Somatic embryos at these different stages of development were collected from in vitro-grown cultures, and then macerated in liquid nitrogen. Proteins were extracted with phenol and further quantified using the Bradford method. The bidimensional electrophoresis analysis revealed a wide range of proteins ranging between 10 and 160 kDa and of pH values ranging from 3 to 10. Several differentially expressed proteins were identified by mass spectrometry, and some were found to be specific to these different stages of somatic embryogenesis in coffee. The enolase and 11S storage globulin proteins, for example, could be used as molecular markers for somatic embryo development stages and for embryogenic and non-embryogenic genotype differentiation, respectively.