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In this study, the optimal hydrogen (H 2) production conditions of the high-efficiency H 2 -producing mutant strain Ethanoligenens harbinense YR-3 (carbon-nitrogen ratio 5.5, phosphate buffer 80 mM, initial pH 6.0, biotin 1.4 mg/L) are obtained by intermittent experiments. The maximum specific H 2 production rate of YR-3 (2.85 mol H 2 /mol glucose) was 1.4 times that of the wild strain ZGX4 (2.04 mol H 2 /mol glucose). The liquid-phase products are mainly ethanol and acetic acid, indicating that the metabolic pathway has not changed. Two-dimensional electrophoresis and mass spectrometry were used to compare and analyze the protein map differences between YR-3 and ZGX4. The results show that 1,6-fructose diphosphate aldolase and the flavoprotein in hydrogenase are highly expressed. This study will provide a theoretical basis for the genetic modification of high-efficiency H 2 -producing strains and the improvement of H 2 production capacity. [ABSTRACT FROM AUTHOR]

In this study, a comprehensive investigation was undertaken to elucidate a simple triazole compound, 5-phenyl-1-(p-tolyl)-1 H-1,2,3-triazole (PPTT), its interactions with high-abundant proteins and identification of low-abundant proteins by serum proteomics. Employing a combination of spectroscopic techniques and computational chemistry, the interactions between PPTT and three high-abundance blood globular proteins, namely human serum albumin (HSA), human immunoglobulin G (HIgG), and hemoglobin (BHb), were explored, thereby ascertaining their binding constants and thermodynamic parameters at the molecular level. Subsequently, based on the differential proteomics, utilizing two-dimensional gel electrophoresis (2-DE) in conjunction with matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS), the research team isolated and identified differentially expressed low-abundance proteins in human blood serum samples following exposure to PPTT. The results showed that there were twenty highly expressed proteins identified from blood serum samples intervened by PPTT. Combining bioinformatics techniques, these proteins were classified, providing preliminary insights like preproprotein or precursors inhibiting the activity of elastase, defending and regulating the immune system, carrying lipid, and other functions into their biological functionalities. One of the differential proteins, apolipoprotein A-1 (ApoA-1) protein, was selected as a possible target to explore the mechanism of action of PPTT intervention on the related signaling pathways involved in human hepatocellular carcinomas(Hep G2) cells. These research findings offer scientifically sound guidance for further in-depth exploration, development, and application of the 1,2,3-triazole compound. [Display omitted] • PPTT has stronger interactions with three high-abundant proteins in blood. • Utilizing serum proteomics to exploit possible biomarkers related to PPTT. • ApoA-1 may be a target of PPTT on the cholesterol metabolism of Hep G2 cells. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is associated with cognitive deficits in humans and animal models. These deficits are paralleled by neurophysiological and structural changes in the central nervous system, particularly in the hippocampus, which plays an important role in memory formation. We previously reported that the magnitude of long-term potentiation at hippocampal Schaffer collateral-CA1 synapses was significantly impaired in streptozotocin (STZ)-induced type 1 diabetic rats (STZ rats). The present study investigated the mechanisms underlying morphological changes in the hippocampus of STZ rats. We performed a proteomic analysis of the hippocampus of STZ rats using two-dimensional gel electrophoresis followed by mass spectrometry. The distribution of 14–3-3 proteins identified by the proteomic analysis was then examined using immunohistochemistry. The results obtained revealed that 14–3-3 η immunoreactivity in the dorsal hippocampus was weaker in STZ rats than in age-matched control rats. Moreover, the density of glial fibrillary acidic protein-immunoreactive astrocytes in the dorsal hippocampus of STZ rats was increased, whereas 14–3-3 η immunoreactivity in astrocytes and neurons in the dentate gyrus was significantly decreased. These results suggest that changes in 14–3-3 η expression are involved in hippocampal astrogliosis or/and neurogenesis in STZ rats. [ABSTRACT FROM AUTHOR]

• Serum A1AT, LRG-1, and clusterin were significantly elevated in IgG4-related disease. • LRG-1 levels were halved after 1 month of prednisolone treatment. • LRG-1 could serve as a novel biomarker of IgG4-related diseases. Immunoglobulin 4 (IgG4)-related disease (IgG4-RD) is a lymphoproliferative disorder characterized by elevated serum IgG4 levels and tissue infiltration of IgG4-positive plasma cells. We analyzed the serum proteins, whose levels varied based on the disease state and treatment. Serum proteins from patients with IgG4-related disease and healthy subjects were resolved using two-dimensional electrophoresis, silver-stained, and scanned. Alternatively, the proteins were labeled with Cy2, Cy3, and Cy5 before electrophoresis. The proteins, whose expression differed significantly between patients and healthy individuals, and between before and after steroid treatment, were identified and validated using enzyme-linked immunosorbent assays. Pre-treatment sera from patients with IgG4-related disease was characterized by increased levels of immunoglobulins such as IgG1, IgG4; inflammatory factors such as α-1 antitrypsin (A1AT); and proteins associated with immune system regulation such as clusterin and leucine-rich α-2-glycoprotein (LRG-1). The serum levels of A1AT, LRG-1 and clusterin, during treatment with prednisolone for up to 12 months revealed that LRG-1 levels were halved after 1 month of treatment, comparable to those in healthy subjects; LRG-1 levels remained normal until the end of treatment. LRG-1 could serve as a novel biomarker of IgG4-related diseases. [ABSTRACT FROM AUTHOR]

The molecular understanding of the pathophysiological changes elicited by diabetes in platelets may help in further elucidating the involvement of this pseudo-cell in the increased risk of developing cardiovascular disease and thrombosis in diabetic subjects. We aimed to investigate the differential characteristics of platelets from diabetic patients and nondiabetic controls to unveil the molecular mechanisms behind the increased platelet reactivity in diabetes. We compared platelets from diabetic and control subjects by 2 dimensional-electrophoresis followed by mass spectrometry. Changes in selected differential proteins were validated by immunoprecipitation assays and western blot. Platelet aggregation was measured by light transmittance aggregometry induced by collagen and ADP, and dynamic coagulation analysis of whole blood was measured by thromboelastometry. We observed significant differences in proteins related to platelet aggregation, cell migration, and cell homeostasis. Subjects with diabetes showed higher platelet aggregation and thrombogenicity and higher contents of the stress-related protein complex HSPA8/Hsp90/CSK2α than nondiabetic subjects. Changes in the chaperones HSPA8 and Hsp90, and in CSK2α protein contents correlated with changes in platelet aggregation and blood coagulation activity. In conclusion, the complex HSPA8/Hsp90/CSK2α is involved in diabetes-related platelet hyperreactivity. The role of the HSPA8/Hsp90/CSK2α complex may become a molecular target for the development of future preventive and therapeutic strategies for platelet dysfunction associated with diabetes and its complications. [ABSTRACT FROM AUTHOR]

Uncovering the genetic basis of hypoxic adaptation is one of the most active research areas in evolutionary biology. Among air-breathing vertebrates, modifications of hemoglobin (Hb) play a pivotal role in mediating an adaptive response to high-altitude hypoxia. However, the relative contributions in water-breathing organisms are still unclear. Here, we tested the Hb concentration of fish at different altitudes. All species showed species-specific Hb concentration, which has a non-positive correlation with altitude. Moreover, we investigated the expression of Hb genes by the RNA-seq and quantitative real-time PCR (qRT-PCR), and Hb composition by two-dimensional electrophoresis (2-DE). The results showed that the multiple Hb genes and isoforms are co-expressed in schizothoracinae fishes endemic to the Qinghai-Tibetan Plateau (QTP). Phylogenetic analyses of Hb genes indicated that the evolutionary relationships are not easily reconciled with the organismal phylogeny. Furthermore, evidence of positive selection was found in the Hb genes of schizothoracinae fishes through the selection pressure analysis. We demonstrated that positively selected sites likely facilitated the functional divergence of Hb isoforms. Taken together, this study indicated that the long-term maintenance of high Hb concentration may be a disadvantage for physiologically acclimating to high altitude hypoxia. Meanwhile, the genetically based modification of Hb-O 2 affinity in schizothoracinae fishes might facilitate the evolutionary adaptation to Tibetan aqueous environments. [ABSTRACT FROM AUTHOR]

• Inherited deficiencies in Golgi proton pump or trafficking proteins can be associated with congenital disorders of glycosylation (CDG). • The gold standard for CDG screening is transferrin isoelectric focusing. • CDG with Golgi homeostasis disruption are associated with abnormal glycosylation profiles of apolipoprotein C-III (apoC-III). • We report three CDG cases with Golgi homeostasis disruption (ATP6V0A2-CDG and COG4-CDG) and normal transferrin glycoforms patterns. • We show the potential of two-dimensional electrophoresis of apoC-III in diagnosing this type of CDG. We identified three cases of congenital disorders of glycosylation (CDG) with Golgi homeostasis disruption, one ATP6V0A2-CDG and two COG4-CDG, with normal transferrin screening analyses. Patient 1 (P1) presented at birth with cutis laxa. Patient 2 (P2) and patient 3 (P3) are adult siblings and presented with severe symptoms evocative of inborn errors of metabolism. Targeted gene sequencing in P1 revealed pathogenic ATP6V0A2 variants, shared by her affected older brother. In P2 and P3, whole exome sequencing revealed a homozygous COG4 variant of unknown significance. In all affected individuals, transferrin analysis was normal. Mass-spectrometry based serum N-glycome analysis and two-dimensional electrophoresis (2-DE) of haptoglobin and of mucin core 1 O-glycosylated apolipoprotein C-III (apoC-III) were performed. All results of second-line N-glycosylation analyses were initially normal. However, apoC-III 2-DE revealed characteristic "apoC-III 1 " pattern in P1 and specific "apoC-III 0 " patterns in P2 and P3. In P2 and P3, this allowed reclassifying the variant as likely pathogenic according to ACMG guidelines. These cases highlight the existence of normal transferrin patterns in CDG with Golgi homeostasis disruption, putting the clinicians at risk of misdiagnosing patients. Furthermore, they show the potential of apoC-III 2-DE in diagnosing this type of CDG, with highly specific patterns in COG-CDG. [ABSTRACT FROM AUTHOR]

The construction of protein abundance profiles helps to interpret the clinical applications of stem cells. Dental pulp stem cells (DPSCs) and gingival mesenchymal stem cells (GMSCs) can be isolated from teeth and used as a highly convenient clinical potential material. Here, we aimed to explore commonalities and differences of DPSCs and GMSCs at the protein level. TMT-based quantitative proteomics and two-dimensional gel electrophoresis technology were used in combination to describe the protein profile of DPSCs and GMSCs extracted from the same donor. A total of 2821 proteins were identified by LC-MS/MS, of which 248 differentially abundant proteins (DAPs) were highly expressed in GMSCs while 782 proteins were highly expressed in DPSCs. The biological functions and molecular pathways of DAPs were annotated with GO enrichment and KEGG analysis. The relationship between molecular abundance and cell characteristics including source, proliferation, angiogenesis and inflammation were connected by WGCNA. Special markers, including Calreticulin (CALR), Annexin A5 (ANXA5) and Rho GDP dissociation inhibitor alpha (GDIR1), were proposed to distinguish DPSCs from GMSCs. Our results provide a molecular basis for in-depth understanding of the protein composition and special functions of dental stem cells, and promote the potential clinical application. • The protein profile of DPSCs and GMSCs without individual difference was described by TMT-based quantitative proteomics and two-dimensional gel electrophoresis technology. • Annotation and enrichment pathways, which were related to autoimmune diseases and immune responses, were enumerated and extracted. • WGCNA analysis was used to construct the relationship between molecular expression and cell characteristics including source, proliferation, angiogenesis and inflammation. • Special markers, including CALR, ANXA5 and GDIR1, were proposed to distinguish DPSCs and GMSCs. [ABSTRACT FROM AUTHOR]

• Multiple biomarkers are a valid strategy for diagnosing six common cancers in Korea. • The AUC of the models in the test set showed very high accuracy. • The sensitivity of the models for early stage cancer (stage I) was very high. The ultimate goal of cancer screening is to diagnose invasive cancers early, while they are still curable. We aimed to validate the diagnostic value of blood-derived protein biomarkers that we developed for six common cancer in Korea. We have discovered 12 protein biomarkers that are useful in differentiating cancer patients from healthy controls using two-dimensional gel electrophoresis (2-DE), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), and literature review. Cancer patients (stomach, colon, liver, lung, breast, and prostate) and control subjects were collected and tested data sets were used to generate predictive models that identify risk scores for each cancer. The validation study was done in serum samples of an independent patient cohort Receiver operating characteristic (ROC) analyses were conducted to evaluate the diagnostic performance of the biomarker combinations. The AUCs of the model in the test set were 0.971, 0.960, 0.969, 0.942, 0.834, and 0.985 for stomach, colon, liver, lung, breast, and prostate cancer, respectively. Combining multiple tumor and systemic inflammatory biomarkers proved to be a valid strategy in the diagnosis of six common cancers in Korea. Further validation of appropriate screening populations through large-scale clinical trials are warranted. [ABSTRACT FROM AUTHOR]

Because the world's wild fish stocks are limited and the market demand is increasing, fish farming has become an alternative food source and a way to reduce costs for consumers. The sale of farmed as wild fish is a fraudulent practice; it is, therefore, important to find new and alternative tools that can help in the fight against fraud to protect consumers and to ensure food traceability. The proteomic profiles of farmed and wild fish differ. With this study we wanted to identify liver protein markers via two-dimensional electrophoresis that would allow us to distinguish wild from farmed gilthead seabream. The liver samples from 32 gilthead seabream, wild and farmed, were stored at −80°C before protein extraction. The samples were subjected to two-dimensional electrophoresis to detect qualitative and quantitative differences. Proteomic analysis showed a protein spot (molecular weight of ∼34 kDa and isoelectric point of ∼6.9) only in the samples from the wild gilthead seabream; liquid chromatography–tandem mass spectrometry identified the spot as ubiquitin. Ubiquitin could be a valid marker to differentiate wild from farmed gilthead seabream; it could be used to ensure continuous monitoring throughout the entire commercial chain and to fight commercial fraud. A proteomic approach to differentiate the method of fish production is proposed. A 2-DE spot, characterized by MS/MS as ubiquitin, differentiated between samples. Liver proteome could be useful to find biological markers. [ABSTRACT FROM AUTHOR]

Drought is the most important abiotic stress limiting wheat production worldwide. Triticum boeoticum , as wild wheat, is a rich gene pool for breeding for drought stress tolerance. In this study, to identify the most drought-tolerant and susceptible genotypes, ten T. boeoticum accessions were evaluated under non-stress and drought-stress conditions for two years. Among the studied traits, water-use efficiency (WUE) was suggested as the most important trait to identify drought-tolerant genotypes. According to the desirable and undesirable areas of the bi-plot, Tb5 and Tb6 genotypes were less and more affected by drought stress, respectively. Therefore, their flag-leaves proteins were used for two-dimensional gel electrophoresis. While, Tb5 contained a high amount of yield, yield components, and WUE, Tb6 had higher levels of water use, phenological related traits, and root related characters. Of the 235 spots found in the studied accessions, 14 spots (11 and 3 spots of Tb5 and Tb6, respectively) were selected for sequencing. Of these 14 spots, 9 and 5 spots were upregulated and downregulated, respectively. The identified proteins were grouped into six functional protein clusters, which were mainly involved in photosynthesis (36%), carbohydrate metabolism (29%), chaperone (7%), oxidation and reduction (7%), lipid metabolism and biological properties of the membrane (7%) and unknown function (14%). We report for the first time that MICP, in the group of lipid metabolism proteins, was significantly changed into wild wheat in response to drought stress. Maybe, the present-identified proteins could play an important role to understand the molecular pathways of wheat drought tolerance. We believe comparing and evaluating the similarity-identified proteins of T. boeoticum with the previously identified proteins of Aegilops tauschii, can provide a new direction to improve wheat tolerance to drought stress. • Of the 14 identified index spots, 9 and 5 spots were upregulated, and downregulated, under drought stress, respectively. • The identified proteins were mainly involved in photosynthesis process. • MICP protein, in the group of lipid metabolism proteins, was significantly changed in wild relatives of wheat (T. boeoticum) in response to drought stress. • MICP protein maybe play an important role to understand the molecular pathways of wheat drought tolerance genotypes. • While, the tolerant genotype contained a high amount of yield, yield components, and WUE; the susceptible genotype had higher levels of water use, phenological related traits, and belowground related characters. [ABSTRACT FROM AUTHOR]

Glycoproteins are important biomarkers for cancers, while most glycoproteomics biomarkers suffering from low sensitivity and specificity due to their uncharacterized glycan structures. AZGP1 is a potential biomarker for salivary diagnostics of lung cancer, which is used as a model glycoprotein in this study for method development. We initially analyzed salivary N-glycoproteome by using lectin affinity chromatography and more than 300 N-glycoproteins were identified, including AZGP1. 7 gel spots of AZGP1 were resolved by two-dimensional gel electrophoresis and further confirmed by two-dimensional western blot as well as mass spectrometry. The isomeric glycan structures of AZGP1 in these spots were systematically characterized both at composition level and at structure level. Our results revealed 10 glycan compositions for salivary AZGP1, including core fucosylated glycans on Asn128 and sialylated glycans on Asn109 and Asn112. We further compared the glycan structures of salivary AZGP1 from lung cancer group and control group. Accordingly, 14 and 7 potential glycan structures were successfully revealed, respectively. In total, 15 glycan compositions and 22 potential glycan structures were identified and characterized for AZGP1, including some different structures with the same compositions. In particular, 5 potential glycan structures were identified as lung cancer unique signatures. Our developed strategy holds promise for thorough identification of glycan structures on a target glycoprotein biomarker. In-depth characterization of its glycan structures will ultimately enhance its sensitivity and specificity for cancer detection. Image 1 • Affinity chromatography and 2-DE were combined for the separation of glycoproteins. • Robust strategy was developed for glycan identification of target proteomic marker. • 15 glycan compositions and 22 glycan structures were characterized for saliva AZGP1. • Unique glycan structures of saliva AZGP1 were revealed for lung cancer. [ABSTRACT FROM AUTHOR]

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Characterization of the uterine proteome before the entry of the conceptus to the uterus is essential to know the factors involved in the physiological events of gestation. The objective of the study was to compare proteomic profile of uterine fluid collected on day 5 post ovulation of cyclic and inseminated mares. Samples of endometrial secretion were recovered over 2 cycles during the fifth day post ovulation. The first cycle constituted the Cyclic group and in the following cycle, the same mares were inseminated and considered as the Inseminated group. All the samples were subjected to two-dimensional electrophoresis (2D-PAGE). A total of 107 spots were visualized by 2D-PAGE. Three spots with differences in abundance between the inseminated and cyclic mares and with presence in at least 80% in one of the groups were selected and identified. The selected spots were extracted, digested by trypsin and analyzed by matrix assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS) for protein identification. Three proteins were identified: ceruloplasmin (CP) serotransferrin (TF) and albumin (ALB). The identified proteins in this study were related to iron metabolism and immunological tolerance suggesting that changes in their abundance during the 5 days after ovulation are probably a signaling mechanism of the potential equine conceptus to the maternal immune system for its immunological recognition. Probably changes in abundance of CP, ALB and TF represent a mechanism of endometrial preparation for the maternal recognition, attachment and development of a potential equine embryo. There is also evidence to support an alternative hypothesis suggesting that protein changes are inflammatory events, resulting from a previous inflammation due to residual seminal effects. • Ceruloplasmin was more abundant in inseminated mares than in cyclic on day 5. • Serotransferrin was more abundant in cyclic mares than inseminated on day 5. • Albumin was more abundant in cyclic mares than inseminated on day 5. • Changes occurred in endometrium fluid before the entry of the embryo into the uterus. [ABSTRACT FROM AUTHOR]

Mucopolysaccharindosis type II (MPS II) is a rare lysosomal storage disorder caused by deficient or absent activity of the iduronate-2-sulfatase (IDS) enzyme, which leads to pathological accumulation of the glycosaminoglycans(GAGs). The absence of early diagnosis can result in irreversible developmental, neurological, and physiological damage. The lack of clear understanding of the etiology of physiological dysfunction in MPS II has been a major obstacle to the development of new treatment. Therefore, a reliable biomarker for early diagnosis and exploration of pathogenic mechanism are of great importance. Proteomics provides powerful tool for protein expression alterations and study of complicated pathological process. This study was performed to identify the differential protein profile in urine of MPS II patients using two-dimensional gel electrophoresis(2D-PAGE)combining with MALDI-TOF/TOF and a total of 15 differentially expressed proteins were identified. Content of alpha1-antitrypsin, Gm2 activator and lipocalin-type prostaglandin D synthase was measured by ELISA method. The value of urinary α1-AT/Cr in MPS II group was 0.79 ± 0.10 mg/mmol, significantly higher than 0.42 ± 0.05 mg/mmol in healthy control group; whereas the value of GM2A/Cr and L-PGDS/Cr in MPS II group was 1.30 ± 0.12 μg/mmol and 9.86 ± 1.16 ng/mmol respectively, which was significantly lower than 2.19 ± 0.19 μg/mmol and 13.98 ± 1.48 ng/mmol in healthy control group. The proteins can be considered as accessory diagnostic biomarkers for MPS II. This approach helped to discover early diagnostic markers and provided a better understanding of the pathogenic mechanism of MPS II. • Proteomic analysis was performed to identify protein profile in urines of MPS II patients. • Expression of proteins changes significantly in urine of MPS II patients. • The differential proteins might provide a non-invasive method to diagnose MPS II. [ABSTRACT FROM AUTHOR]

• Neonatal polyI:C treatment caused behavioral abnormalities in adulthood. • Antipsychotics ameliorated behavioral abnormalities in neonatal polyI:C-treated mice. • Proteomic analysis revealed the changes in ALDH1L1 and CRMP5. Perinatal virus infection is an environmental risk factor for neurodevelopmental disorders such as schizophrenia. We previously demonstrated that neonatal treatment with a viral mimetic, polyriboinosinic-polyribocytidilic acid (polyI:C), in mice leads to emotional and cognitive deficits in adolescence. Here, we investigated the effects of antipsychotics on polyI:C-induced behavioral abnormalities. We also performed a proteomic analysis in the hippocampus of polyI:C-treated adult mice using two-dimensional electrophoresis to understand the changes in protein expression following neonatal immune activation. Neonatal mice were subcutaneously injected with polyI:C for 5 days (postnatal day 2–6). At 10 weeks, sensorimotor gating, emotional and cognitive function were analyzed in behavioral tests. Clozapine improved PPI deficit and emotional and cognitive dysfunction in polyI:C-treated mice. However, haloperidol improved only PPI deficit. Proteomic analysis revealed that two candidate proteins were obtained in the hippocampus of polyI:C-treated mice, including aldehyde dehydrogenase family 1 member L1 (ALDH1L1) and collapsin response mediator protein 5 (CRMP5). These data suggest that the neonatal polyI:C-treated mouse model may be useful for evaluating antipsychotic activity of compounds. Moreover, changes in the protein expression of ALDH1L1 and CRMP5 support our previous findings that astrocyte-neuron interaction plays a role in the pathophysiology of neurodevelopmental disorders induced by neonatal immune activation. [ABSTRACT FROM AUTHOR]

Naja kaouthia is one of the most prevalent medically important snakes of North East India and Bangladesh responsible for most of the bite cases. In this study, an attempt was made to decipher venom variation of Naja kaouthia venom from North East India and Bangladesh. Using multidimensional methods including reverse phase HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional gel electrophoresis (2D-PAGE), the quantitative differences in venom composition have been revealed. Moreover, tested in-vitro biochemical and biological activities also exhibited differences which could be due to venom variability. Furthermore, neutralization efficacy of commercially available Indian polyvalent antivenoms (Vins, Bharat Serum, Haffkine) was evaluated and the results displayed significant differences in neutralizing efficacy between the antivenoms. Immunoblotting experiments showed antivenom molecules cross reacted with high molecular mass components while poorly reacted towards low molecular mass proteins. Immuno-depletion study demonstrated that Vins polyvalent antivenom was poor in immunocapturing the venom proteins of both North East Indian and Bangladesh origin Naja kaouthia at the ratio of 1:16 (venom: antivenom). Image 1 • North East Indian and Bangladesh origin Naja kaouthia venom demonstrates variation. • Neutralization of venom toxic effects was analysed using three Indian polyvalent antivenoms. • Haffkeine polyvalent antivenom performed better in neutralizing toxic effects compared to Vins and Bharat Serum. • Antivenomics study revealed poor immunocapturing ability of Naja kaouthia venom proteins by Vins polyvalent antivenom. [ABSTRACT FROM AUTHOR]

Abstract Bombyx mori nuclear polyhedrosis virus (BmNPV) is the most damaging virus for the production of silkworm cocoons. Antivirus research continues to be an important aspect of the silkworm industry. Two-dimensional electrophoresis and mass spectrometry have been applied for analyzing the midgut proteome of BmNPV-infected silkworms. In recent years, the isobaric tags for relative and absolute quantitation (iTRAQ) method has frequently been used when studying interaction between BmNPV and Bombyx mori , and useful information has been obtained. In this study, midgut proteins of BmNPV-infected silkworms were extracted from silkworm variety NIL·LVR with anti-BmNPV activity at 48 h, and proteome analysis was carried out using the label-free method. 2196 proteins were identified. Among them, there were 85 differentially expressed proteins, 45 upregulated proteins (immune-activated proteins), 28 downregulated proteins, and six proteins were specific for the BmNPV group and another six specific for control group. Many of the immune-activated proteins have been reported to have innate immune functions, and the downregulated proteins are involved in apoptosis or abnormal cell viability. In conclusion, this study provides evidence for host defense against BmNPV infection by both innate immunity and apoptosis, revealing the potential function of the midgut after oral infection of BmNPV in Bombyx mori. Significance Bombyx mori nuclear polyhedrosis virus (BmNPV) has a great impact on the sericulture industry. However, the mechanism of resistance to BmNPV has not been fully elucidated. The silkworm midgut is not only the major organ for food digestion and nutrient absorption but also an immune organ serving as the first line of defense against microbial invasion and proliferation. Here we combined label-free quantitative proteomic, bioinformatics, quantitative real-time PCR and SDS-PAGE analyses and found that BmNPV invasion causes complex protein alterations in the larval midgut of NIL·LVR with anti-BmNPV activity. The results showed that many upregulated differentially expressed proteins have been reported to have innate immune functions and the downregulation proteins are involved in apoptosis or abnormal cell viability. These findings provide evidence for host defense against BmNPV infection by both innate immunity and apoptosis, and reveals the potential function of the midgut after infection of BmNPV in Bombyx mori. Graphical abstract Unlabelled Image Highlights • Quantitative proteomics of the silkworm midgut after BmNPV infected by label-free method • The research material is antiviral NIL•LVR strain. • Among the 85 differentially expressed proteins identified, six were in the BmNPV group and six were in the control group. • Some key differentially expressed proteins were validated by quantitative real-time PCR. • These data reveals the potential function of the midgut after oral infection of BmNPV in Bombyx mori. [ABSTRACT FROM AUTHOR]

Abstract Trees dominate the structural and functional dynamics of many temperate and tropical forest ecosystems and are of considerable scientific and social interest. The effective ecological restoration of abandoned agricultural fields, especially of highly degraded ecosystems, remains a challenge. Germination is imperative to restore natural ecosystems and to save the environment. Low germination rate is key player to disturb the ecosystem. Cyclobalnopsis gilva is an economically important woody plant, however its germination rate is less than 50% in its natural habitats compared to that of other plants. A comparative proteomics approach was carried out to investigate this feature on germinated and non-germinated seeds of C. gilva. Proteins from seeds of C. gilva were extracted using phenol extraction, separated by two-dimensional electrophoresis, and identified through matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. In addition, the results of proteome were verified through the RT-qPCR determination. More than 700 proteins were reproducibly detected. Among 26 proteins with 2-fold changes in abundance, the 24 differential proteins were identified successfully. Many differential proteins were involved in starch metabolism. β-amylase abundance and mRNA level were both up-regulated in germinated seeds of C. gilva. An important point to provides new insights into the understandings of C. gilva seed germination problems are found through 2-DE and RT-qPCR analyses to save the forest ecology and solve the problem of woody plants with low germination rate all over the world. Highlights • Cyclobalnopsis gilva is an economically important woody plant with low seed germination rate. • A comparative proteomics and molecular approach was carried out to investigate low germination of C. gilva seeds. • Low seed germination study of C. gilva provides an insight that could be important for other woody plants. [ABSTRACT FROM AUTHOR]

The genes encoding the ribosomal RNA (rRNA) subunits of the amoeba Naegleria gruberi are encoded in a relatively uncommon arrangement: on a circular extrachromosomal DNA element with each organism carrying about 4,000 copies of the element. As complete sequence analysis of the N. gruberi chromosomal DNA revealed no copy of the rRNA genes, these extrachromosomal elements must therefore replicate autonomously. We reported elsewhere the molecular cloning and the complete sequence analysis of the entire rRNA gene-containing element of N. gruberi (strain EG B). Using neutral/neutral two-dimensional agarose electrophoresis, the region in the element enclosing the single replication origin using DNA from asynchronous and axenically propagated N. gruberi populations was localized within a 2.1 kbp fragment located approximately 2,300 bp from the 18 S rRNA gene and 3,700 bp from the 28 S rRNA gene. The results indicate that replication occurs from a single origin via a theta -type mode of replication rather than by a rolling circle mode. Further, G-quadruplex elements, often located near DNA replication origins, occur in and near this fragment in a repeated sequence. [ABSTRACT FROM AUTHOR]

Highlights • Proteomics highlights the central metabolism roles in acid tolerance of R. freirei. • An integrated model is proposed based on identified proteins. • Identified proteins reveals an inclination towards a catabolic state. • A crosstalk between acid and oxidative stress is suggested. Abstract Soil acidity strongly affects microbial diversity and represents a constraint to legume production. Rhizobium freirei is a common bean symbiont recognized by its tolerance to environmental stresses, including low pH. The protein expression profiles of R. freirei PRF 81T grown in pH 6.8 and 4.8 were analyzed to clarify the mechanisms responsible for acid tolerance in this species. Bacteria were grown up to exponential phase in tryptone-yeast (TY) medium at pH 6.8 and 4.8. Whole-cell protein extracts were separated by two-dimensional electrophoresis (2-DE), and spots that showed statistical difference in their relative volumes (%vol) between the treatments were selected and excised for identification by MALDI-TOF mass spectrometry. Data showed that protein synthesis was increased at pH 4.8, which consequently raises energy demand. Differential expression of membrane-associated proteins suggested an increased proton extrusion and decreased influx, while central metabolism seemed to be through inducing acid-consuming catabolic pathways and preventing fatty acid biosynthesis. Consequently, the respiratory chain was stimulated along with the production of reactive oxygen species (ROS). The broad range of metabolic pathways modulated by acidified pH endorses the adaptive response to environmental stresses as a multigenic character in R. freirei PRF 81. Moreover, our data emphasize the key role of central metabolism in acid stress tolerance. [ABSTRACT FROM AUTHOR]

Abstract Premature ovarian failure (POF) is defined when a female achieves menopause before the age of 40. Although many conditions are known to be causative for POF, the most common one is idiopathic. This study was undertaken to investigate the pathogenesis of POF using proteomic tools. Two-dimensional electrophoresis (2-DE) analysis was performed to screen for proteins differentially expressed in patients with POF. Using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we identified 11 significant proteins differentially expressed in the serum of POF patients: 5 proteins with expression increased more than two folds, 5 proteins with expression decreased more than two folds, and 1 protein expressed specifically in the serum of patients with POF. The results of the 2-DE analysis were further validated by Western blotting and ELISA analyses, which 5 reproductive system-related proteins (Ceruloplasmin, Complement C3, Fibrinogen α, Fibrinogen β, and SHBG) were selected. The different expression levels for these proteins were confirmed and demonstrated the possibility of using them as biomarkers to screen POF. These pre-clinical data provide plausible translational implications for targeting the pathogenesis of POF for each protein. Highlight • Ceruloplasmin, Complement C3, Fibrinogen α, Fibrinogen β, and SHBG were differentially expressed in POF patients. • They can be used as provisional biomarkers for identifying POF patients. • These proteins may be helpful in understanding the molecular mechanism of the causes, pathogenesis, and management of POF. • This is the first identification of differentially expressed proteins in the serum of POF patients. [ABSTRACT FROM AUTHOR]

Abstract There is a complex cascade involving proteins during early embryo development and maternal recognition, which is very important for maintenance of a conceptus. The aim of this study was to compare proteomic profile of uterine fluid after ovulation in pregnant and cyclic mares. In the first cycle, samples of uterine fluid of 30 cyclic mares were collected on days 7 (n = 10), 10 (n = 10) and 13 (n = 10) post ovulation and constituted the Cyclic group. In the second cycle, the same mares were bred to a fertile stallion. At days 7, 10 and 13 uterine fluid samples were collected. Immediately after sample collection, the mare's uteri were flushed, and those with an embryo recovered were assigned to the Pregnant group. Of the 30 mares flushed embryos were recovered from 6 mares on day 7, 6 on day 10 and 6 on day 13. Samples from the mares without embryo recovery were excluded from both groups. The uterine fluid samples were processed by two-dimensional electrophoresis technique followed by matrix assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry for the identification of relevant protein spots. From a total of 677 detected spots 19 were identified, 13 more abundant in Pregnant group and 6 in Cyclic group. In summary, pregnant and cyclic mares showed proteins with different abundance. Identified proteins were related to the transport of lipids through the embryo capsule, uterine motility, ATP generation, maternal immunological tolerance, cell proliferation, differentiation, metabolism and angiogenesis. Changes in the proteomic profile of uterine fluid during early embryo development in mares were related with the conceptus presence, suggesting that these alterations may be important for conceptus development and maternal recognition of pregnancy. Highlights • Pregnant and cyclic mares showed proteins with different abundance. • Changes in the proteomic profile were related with the embryonic presence. • Proteins were related to the transport of lipids through the embryo capsule. • Identified proteins were related to uterine motility and ATP generation. • Proteins were related to angiogenesis and maternal immunological tolerance. [ABSTRACT FROM AUTHOR]

Abstract In this work, we explored the "deep" seed peanut proteome by using both two dimensional electrophoresis (2-DE)-based analysis run under reducing and non-reducing condition (protein-centric) and LC-MS/MS gel-free proteomic (peptide-centric). The former approach allowed to identify high molecular weight disulfide-linked Ara h 1 and Ara h 3 heteroligomers and Ara h 1 homoligomers linked through covalent bonds other than disulfides. The occurrence of these protein complexes revealed natural interactions between Ara(s) subunits with a possible involvement in the allergenic potential of peanut. The second approach, also referred to as shot-gun proteomics, allowed the identification of 149 gene products, including low-abundance proteins escaped the 2-DE detection. Interestingly, we identified 60 proteins never catalogued previously. The complementary exploitation of two proteomic approaches enabled the access to new relevant information about the complexity of the peanut proteome, with special emphasis to the complement of allergens (allergome). Graphical abstract Unlabelled Image Highlights • High MW disulfide-linked multimers of Ara h 1 and Ara h 3 were characterized. • Ara h 1 oligomers aggregated via covalent bonds other than disulfides. • 2DE and shot-gun proteomics were complementary to resolve the peanut allergome. • Identification of 149 peanut seed gene products. [ABSTRACT FROM AUTHOR]

Abstract The main intricacy in the human proteome is that it is tremendously complex and composed from diverse and heterogeneous gene products. These products are called protein species or proteoforms and are the smallest units of the proteome. In pursuit of the comprehensive profiling of the human proteome, significant advances in the technology of so called "Top-Down" mass spectrometry based proteomics, have been made. However, the scale of performance of this approach is still far behind the "Bottom-Up", peptide-centric techniques. The classical two-dimensional electrophoresis (2-DE) as the most powerful and convenient method for separation of proteoforms remains as a superior method in "Top-Down" proteomics. Here, some aspects of approaches for establishing an inventory of proteoforms based on 2-DE and mass spectrometry are discussed. Biological significance The systematic efforts in the Human Proteome project to map the entire human proteome greatly depend on currently available and emerging techniques and approaches. Here, the possibilities of a visual representation of the human proteome by combination of virtual/experimental 2-DE with protein identification by mass spectrometry or immunologically is discussed. By application of this approach on several profiles of gene products we show its convenience in informative representation of the whole proteome and single gene products, proteoforms (protein species). This approach could be very helpful in the emerging global inventory of all human proteoforms. Graphical abstract Unlabelled Image Highlights • There is need of development of a knowledge base for the inventory of human proteoforms • Some approaches for obtaining and establishing an inventory of information about proteoforms are discussed. • It is shown that 2-DE methodology together with mass spectrometry can be a good complement for proteoforms atlas [ABSTRACT FROM AUTHOR]

Abstract Fibromyalgia (FM) is a chronic pain disorder characterized by widespread pain and associated with unspecific symptoms. So far, no laboratory tests have been validated. The aim of the present study was to investigate the presence in saliva of potential diagnostic and/or prognostic biomarkers which could be useful for the management of FM patients. Specifically, the salivary profile of FM patients was compared with those of healthy subjects, subjects suffering migraine (model of non-inflammatory chronic pain), and patients affected by rheumatoid arthritis (model of inflammatory chronic pain). For proteomics analysis 2-DE and SELDI-TOF-MS were applied. From 2-DE serotransferrin and alpha-enolase were found differentially expressed in FM. Hence, their expression was validated by ELISA together with phosphoglycerate-mutase-I and transaldolase, which were found in a previous work. Moreover, ROC curve was calculated by comparing FM patients versus control subjects (healthy plus migraine) to investigate the discriminative power of biomarkers. The best performance was obtained by combining alpha-enolase, phosphoglycerate-mutase-I and serotransferrin. On the other hand, none of the candidate proteins showed a statistical correlation with clinical features. Finally, preliminary SELDI analysis highlighted two peaks whose identification need to be validated. Overall, these results could be useful in supporting the clinical diagnosis of FM. Significance FM is one of the most common chronic pain condition which is associated with significant disability. The fibromyalgic pain is a peculiar characteristic of this disease and FM patients suffer from reduced quality of life, daily functioning and productivity. Considering the deep complexity of FM, the discovery of more objective markers is crucial for supporting clinical diagnosis. Therefore, the aim of the present study was the selection of biomarkers effectively associated with fibromyalgic pain which will enable clinicians to achieve an unambiguous diagnosis, and to improve approaches to patients' management. We defined a panel of 3 salivary proteins which could be one of the criteria to be taken into account. Consequently, the identification of disease salivary biomarkers could be helpful in detecting FM clusters and targeted treatment. Actually, our future perspective foresees to develop a simple, rapid and not invasive point-of-care testing which will be of use during the diagnostic process. In addition, the present results can offer a clue for shedding light upon the complex entity of such a disease like FM. Graphical abstract Unlabelled Image Highlights • The diagnosis of FM basically relies on the clinicians experience and no laboratory tests have been validated. • We investigated the presence in saliva of eventual diagnostic and/or prognostic biomarkers useful for the management of FM patients. • A panel combing 3 proteins, ENOA, PGAM1and TRFE, is proposed as an useful tool in supporting clinicians in diagnosis of FM. [ABSTRACT FROM AUTHOR]

Abstract Objective We investigated effects of salazosulfapyridine (SASP) on the protein profile of cell surface (CS)-proteins of SW982, a human synovial sarcoma cell line, using biotinylation of CS-proteins and 2-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Methods SW982 cells were treated with SASP and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5ASA). Then the cells were treated with a membrane-impermeable biotinylating reagent. Biotinylated CS-proteins were isolated using NeutrAvidin-bound beads. CS-proteins affected by the drugs were detected by 2D-DIGE and subjected to mass spectrometry. Results By the 2D-DIGE analysis, in total 576 spots were detected, 29 out of which showed more than ±1.5-fold different intensity in the SASP-, SP-, and 5ASA-treated cells, compared to non-treated cells (p < 0.05). Interestingly, 7 out of the 29 spots changed their intensity only by SASP and 17 spots changed their intensity only by SP. We identified 9 protein from 15 out of the 29 spots, most of which were evidenced to exist on the cell surface by flow cytometry. Conclusion We found novel effects of SASP and its metabolites on SW982 cells by the combination of biotinylation of cell surface proteins and 2D-DIGE analysis. These data would help understanding of anti-rheumatic actions of SASP. Furthermore, the combination would be a useful method for the analysis of CS-proteins in various conditions. Highlights • Effects of salazosulfapyridine on cell surface proteins were analyzed by 2D-DIGE. • About 1/5 of the detected 576 spots were affected by the drug and its metabolites. • Most of the identified proteins were detected on the cell surface by flow cytometry. [ABSTRACT FROM AUTHOR]

[Display omitted] • The coffee protein profile differs before fermentation and after drying. • 2D-PAGE separation reveals significant differences in spot distribution to control. • Fermentation provides a valuable tool for modifying proteins and volatile compounds. This work aims to analyze the protein profile and volatile compounds of coffees fermented with the indigenous microbiota and with the co-inoculation of three yeasts (Saccharomyces cerevisiae , Torulaspora delbrueckii , and Candida parapsilosis). Two-dimensional gel electrophoresis (2D-PAGE), MALDI-ToF/ToF (MS/MS), and gas chromatography (GC–MS) were performed. A total of 72 "spots" were detected by 2D-PAGE. 16 spots were selected for identification by MALDI-ToF/ToF, and 12 were identified (11S protein, 13S globulin basic chain, 17.6 kDa class II heat shock protein (HSP17.6-CII), 18.0 kDa class I heat shock protein, Seed of Late Development Stage, Pru ar 1, and FAR-1 protein). 81 main volatile compounds were detected and classified into alcohols, acids, aldehydes, esters, hydrocarbons, pyrazines, furans, thiols, and pyridines/pyrrols. The difference between the identified volatile compounds and their concentrations was detected in the treatments with and without inoculation after drying. The compounds formed in green coffee during fermentation can participate in several reactions during roasting, presenting different sensory profiles and contributing to coffee quality. [ABSTRACT FROM AUTHOR]

Abstract Olive is one of the most important fruit crop trees in the history of Mediterranean because of the high quality oil. Olive oil has a well-balanced fatty acid composition along with biophenols, which make it exceptional in human diet and provide an exceptional value to the olive oil. Leaf non-glandular peltate trichomes are specialized cell types representing a protective barrier against acute environmental conditions. To characterize the proteome of this highly differentiated cell type, we performed a comparative proteomic analysis among isolated trichomes and trichome-less leaves. Proteins were separated and identified using the 2-DE MALDI-TOF/MS method. A number of enzymes involved in abiotic and biotic stress responses are present and may be responsible for the adaptation to prolonged adverse environmental conditions. The results show that this highly differentiated cell type is physiologically active fulfilling the demands of the trichomes in furnishing the leaf with a highly protective mechanism. [ABSTRACT FROM AUTHOR]

Abstract Mercury (Hg) is a hazardous chemical in the environment and can accumulate in the food chain. Selenium (Se) is a necessary element for human health and has antagonistic effects on Hg toxicity. In this work, we investigated the effect of Se on Hg containing and Hg-responsive proteins in rice using 1, 2-dimensional electrophoresis combined with SR-XRF techniques. Two weeks old rice seedlings were exposed to Hg and/or Se compounds. After 21 days proteins in the rice roots were separated by electrophoresis and their metal contents were determined by X-ray fluorescence to identify Hg and Se responsive biomolecules. The results show that under Hg stress alone Hg is bound to proteins with molecular weights of 15–25 kDa. With the addition of Se, a new Hg-containing protein band in the 55–70 kDa range was also found, while the content of Hg in the 15–25 kDa proteins decreased. Ten and nine new protein spots were identified after adding Se to inorganic Hg and methylmercury exposed roots, respectively. Adding Se regulates the abundance of proteins associated with carbohydrate and energy metabolism, stress response, cell cycle, and DNA replication indicating that these proteins mediate the antagonism of Se against Hg toxicity. This study helps us to better understand the molecular mechanism of Hg tolerance as well as the molecular antagonism between Hg and Se in rice plants. [ABSTRACT FROM AUTHOR]

Abstract The purpose of this study was to establish a method for determining the bacteriolytic activity after separation of lysozyme-binding proteins from egg white. Lysozyme-binding proteins such as ovotransferrin and ovalbumin were separated by non-denaturing two-dimensional electrophoresis (2DE) and transferred to a membrane. The lysozyme activity of the separated and immobilized egg white proteins was assessed directly to produce a non-denaturing 3D map of the egg white proteins by incorporating an axis that combined each spot's lysozyme-activity with the non-denaturing 2DE pattern. Lysozyme-ovotransferrin and lysozyme-ovalbumin complexes could be reconstructed in vitro after the cathode end fraction containing lysozyme was added to purified ovotransferrin and ovalbumin, respectively. These complexes retained lysozyme activity even after separation by non-denaturing 2DE. Furthermore, when the lysozyme-ovotransferrin complex from egg white was extracted after separation by isoelectric focusing by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, the complex possessed bacteriolytic activity against both Bacillus subtilis and Escherichia coli. These methods can be applied to investigate protein complexes possessing bacteriolytic activity against a wide range of both Gram-positive and Gram-negative bacteria. Highlights • Determining the bacteriolytic activity after separation of lysozyme-binding proteins. • Production of a non-denaturing 3D map of the egg white proteins • Separation and extraction of the lysozyme-ovotransferrin complex from egg white. • The complex possesses bacteriolytic activity against both B. subtilis and E. coli [ABSTRACT FROM AUTHOR]

Abstract Moisture stress is the most important limitation of wheat production in the worldwide. Among the tribe Triticeae , Aegilops tauschii is one of the most valuable gene sources of resistance to abiotic stresses. In order to identify the most tolerant accession to moisture stress, and to understand its adaptive mechanisms at the molecular level, the present experiment was carried out on ten Ae. tauschii accessions under normal (95% soil pot capacity) and moisture stress (45% soil pot capacity) conditions. At the start of the heading time, the expanded flag leaves of treated and untreated plants were sampled for two-dimensional electrophoresis (2-DE) based on proteomics approach. A19 accession was less affected by the imposed moisture stress; therefore, it was used for the proteomics experiment. Among 252 protein spots which were reproducibly detected in each given 2-DE gels, 25 spots showed significant differences between the two moisture treatments; 17 spots were upregulated and 8 spots were downregulated. The identified proteins by MALDI-TOF/TOF, were allocated to seven functional protein groups, which were mainly involved in photosynthesis/respiration (28.5%), carbohydrate metabolism (14.2%), energy metabolism (7.1%), chaperone (14.2%), protein translation and processing (14.2%), repair and stability of the genome (7.1%) and unknown function (14.2%). We report this for the first time that RMI2 protein (in the group of repair and stability of the genome) was significantly changed in wheat in response to moisture stress. We believe that, the identified proteins could play important roles in acclimation and tolerance to moisture stress and provide the genetic pathways for improving tolerance to moisture stress in wheat. [ABSTRACT FROM AUTHOR]

The substitution and sale of frozen-thawed fish labeled as fresh is a widespread, difficult to unmask commercial fraud and a potential risk for consumer health. Proteomics could help to identify markers for the rapid screening of food samples and the identification of frozen-thawed seafood. Using two-dimensional electrophoresis (2-DE) and high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS), we identified biomarkers that are able to discriminate between fresh and frozen-thawed tissue samples of curled octopus ( Eledone cirrhosa) . The 2-DE analysis showed a significant reduction in two protein spots (molecular weight of 45–50 kDa, isoelectric point of 6.5–7) identified as transgelin. At shotgun analysis, nine proteins resulted modulated and transgelin was confirmed as down-regulated, making it a potentially useful marker for differentiating between fresh and frozen-thawed fish product samples. Biological significance This work, based on two different proteomics approaches, investigated differentially expressed proteins in the tentacles of the curled octopus ( E. cirrhosa ) after freezing-thawing processes. We were able to characterize the proteome of the tentacles, increasing our knowledge on this species, and a common down-regulated protein was identified by 2-DE and shotgun analysis, a calponin-like protein called transgelin, suggesting a potential use as a marker to distinguish different states of conservation in this species. [ABSTRACT FROM AUTHOR]

We develop a new two-dimensional (2D) high performance liquid chromatography (HPLC) approach for intact protein analysis. Development of 2D HPLC has a bottleneck problem – limited second-dimension (second-D) separation speed. We solve this problem by incorporating multiple second-D columns to allow several second-D separations to be proceeded in parallel. To demonstrate the feasibility of using this approach for comprehensive protein analysis, we select ion-exchange chromatography as the first-dimension and reverse-phase chromatography as the second-D. We incorporate three second-D columns in an innovative way so that three reverse-phase separations can be performed simultaneously. We test this system for separating both standard proteins and E. coli lysates and achieve baseline resolutions for eleven standard proteins and obtain more than 500 peaks for E. coli lysates. This is an indication that the sample complexities are greatly reduced. We see less than 10 bands when each fraction of the second-D effluents are analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE), compared to hundreds of SDS-PAGE bands as the original sample is analyzed. This approach could potentially be an excellent and general tool for protein analysis. [ABSTRACT FROM AUTHOR]

Male reproductive capacity is essential for animal breeding and reproduction. In males, the testes produce sperm and secrete androgen, processes which require precise regulation by multiple proteins. The composition of proteins in the ram testes has not yet been studied systematically, thus, the application of proteomics to explore differential protein regulation during ram testes development is of great significance. In the present study, ram testes were studied at five different developmental phases to assess postnatal differences in protein regulation. Two dimensional electrophoresis (2-DE) was used to separate ram testes proteins at each developmental phase, yielding 45 different proteins, 37 of which were identified by Matrix Assisted Laser Desorption Ionization-Time of Flight-Time of Flight-Mass Spectrometry (MALDI-TOF/TOF-MS). Gene Ontology (GO) was used to specifically annotate the biological process, cellular composition, and molecular function of each identified protein. Most of the identified proteins were involved in structural formation, development, reproduction, and apoptosis of the testicular spermatogenic tissue and spermatozoa. Quantitative real time PCR (qRT-PCR), western blot and immunohistochemical methods were used to verify the proteins, and the results were consistent with that of 2-DE. The proteins that were different in abundance that were identified in this study can be used as biomarkers in future studies of ram reproduction. [ABSTRACT FROM AUTHOR]

The main objectives of the study were to: (1) deeply analyse the serum protein composition of Equus caballus, (2) assess the effectiveness of the high-abundant protein depletion and improve the concentration of medium- and low-abundant proteins. The analysis were performed on the blood plasma of three healthy part-Arabian mares. The implementation of two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation - time of flight mass spectrometry allowed us to establish a horse plasma proteome map. Serum proteins were resolved at pH 4 to 7, followed by 12% SDS-PAGE. As a result 136 spots were successfully identified, representing the products of 46 unique genes. Of these, 22 gene products have not been previously identified in horse serum/plasma samples using proteomic tools. Gene ontology analysis showed that almost 30% of all identified gene products belong to the coagulation and complement cascades. These results can undoubtedly serve as a useful and prospective prerequisite for the future analysis of horse plasma proteome changes in different physiological and pathophysiological conditions. The use of the medium- and low-abundant protein enrichment tool increased their abundance and allowed us to identify a higher number of protein gene products. The highest depletion efficiency was observed for the most abundant plasma proteins, that is albumin, IgG heavy chains and serotransferrin. [ABSTRACT FROM AUTHOR]

[Display omitted] • Exotoxin A was detected in the hemolymph, hemocytes, and fat body of G. mellonella. • Injection of exoA causes a decrease in the protein concentration in all tissues. • Changes in the protein concentration are correlated with the time of exoA detection. • ExoA influences apoLp-III abundance in the hemocytes and the fat body. • ExoA causes changes in apoLp-III isoforms and derived polypeptides in all tissues. In the present study, we have demonstrated a correlation in time between changes in the amount of apolipophorin III (apoLp-III) in the fat body and hemocytes of Galleria mellonella larvae challenged with Pseudomonas aeruginosa exotoxin A (exoA). An increase in the amount of apoLp-III was detected 1–8 h after the challenge; then, a temporary decrease was observed after 15 h followed by an increase in the level of apoLp-III, however to a different extent. The profile of apoLp-III forms in the hemolymph, hemocytes, and fat body of the exoA-challenged larvae was analyzed using two-dimensional electrophoresis (IEF/SDS-PAGE) and immunoblotting with anti-apoLp-III antibodies. Two apoLp-III forms differing in isoelectric point values estimated at ∼ 6.5 and ∼ 6.1 in the hemolymph and ∼ 6.5 and ∼ 5.9 in the hemocytes as well as one isoform with p I ∼ 6.5 in the fat body with an additional apoLp-III-derived polypeptide with estimated p I ∼ 6.9 were detected in the control insects. The injection of exoA caused a significant decrease in the abundance of both apoLp-III isoforms in the insect hemolymph. In the hemocytes, a decrease in the amount of the p I ∼ 5.9 isoform was detected, while the major apoLp-III isoform (p I ∼ 6.5) remained unchanged. In addition, appearance of an additional apoLp-III-derived polypeptide with an estimated p I ∼ 5.2 was observed. Interestingly, there were no statistically significant differences in the amount of the main isoform in the fat body between the control and exoA-challenged insects, but the polypeptide with p I ∼ 6.9 disappeared completely. It should be noted that the decrease in the amount of apoLp-III and other proteins was especially noticeable at the time points when exoA was detected in the studied tissues. [ABSTRACT FROM AUTHOR]

The functions and properties of fungal sheath, an extracellular polysaccharide produced by many white-rot fungi, have been studied. However, the strong adherence of the sheath to fungal hyphae had been a major impediment in preparing intracellular proteins from the fungi and analyzing their cellular responses. To overcome this issue, we developed a rapid and easy method to remove the polysaccharide sheath using a selective lignin degrader, Ceriporiopsis subvermispora , which produces large sheath amounts in the presence of a lignin-derived aromatic compound. Using this approach, we achieved thorough removal of sheath and cell disruption using beads and a solution with a high protein-solubilizing power, which enabled the efficient extraction of intracellular proteins from C . subvermispora surrounded by sheath. In addition, for proteomic analysis, we investigated whether these extracted proteins were compatible with two-dimensional electrophoresis. By efficiently concentrating on protein solubilization in the first dimension and using a stacking gel in the second dimension, we successfully obtained a high-resolution proteome map of C . subvermispora . We also used the same proteins for fluorescence two-dimensional difference gel electrophoresis to obtain the quantitative protein expression profiles. These steps demonstrated that two-dimensional electrophoresis-based proteomics can be used to clarify the composition of intracellular proteins from sheath-producing white-rot fungi. [ABSTRACT FROM AUTHOR]

Nine geographically diverse Moniliophthora perniciosa (witches' broom disease pathogen) isolates were cultured in vitro . They included six C-biotypes differing in virulence on cacao ( Theobroma cacao ), two S-biotypes (solanaceous hosts), and an L-biotype (liana hosts). Mycelial growth rates and morphologies differed considerably, but no characters were observed to correlate with virulence or biotype. In plant inoculations using basidiospores, one C-biotype caused symptoms on tomato (an S-biotype host), adding to evidence of limited host adaptation in these biotypes. Mycelial proteomes were analysed by two-dimensional gel electrophoresis (2-DE), and 619 gel spots were indexed on all replicate gels of at least one strain. Multivariate analysis of gel spots discriminated the L-biotype, but not the S-biotypes, from the remaining strains. The proteomic similarity of the S- and C-biotypes is consistent with their reported lack of phylogenetic distinction. Sequences from tandem mass spectrometry of tryptic peptides from major 2-DE spots were matched with Moniliophthora genome and transcript sequences on NCBI and WBD Transcriptome Atlas databases. Protein-spot identifications indicated that M. perniciosa saprotrophic mycelial proteomes expressed functions potentially connected with a ‘virulence life-style’, including peroxiredoxin, heat-shock proteins, nitrilase, formate dehydrogenase, a prominent complement of aldo-keto reductases, mannitol-1-phosphate dehydrogenase, and central metabolism enzymes with proposed pathogenesis functions. [ABSTRACT FROM AUTHOR]

Congenital disorders of glycosylation (CDGs) are rare inherited disorders affecting glycosylation of proteins and lipids and sharing very heterogeneous multivisceral symptoms. The biochemical screening of these diseases is currently limited to electrophoresis or HPLC separation/quantification of serum transferrin glycoforms and is relatively frequently hampered by genetic polymorphism. Further, it has been shown that transferrin glycosylation can be very poorly affected in confirmed CDGs. We developed a fast and simple two-dimensional (2-DE) Western-blot analysis applied to the simultaneous detection of various serum glycoproteins, i.e. haptoglobin, α1-anti-trypsin, transferrin and α1-acid glycoprotein, and applied it to a large cohort of CDGs and secondary glycosylation disorders. When separated using 2-DE, haptoglobin β glycoforms showed clear abnormalities in all interpretable CDG type I and CDG type II patterns. Although secondary glycosylation defects such as alcoholism, untreated fructosemia and bacterial neuraminidase remain to be excluded, we showed that 2-DE pattern of haptoglobin β glycoforms thus constitute a very reliable additional biomarker of all types of CDGs. Coupled with common screening techniques and glycans mass spectrometry, it can orientate and facilitate the way towards CDG molecular diagnostic. [ABSTRACT FROM AUTHOR]

Many biochemical events occur inside grains during post-harvest processes. Several methods have been developed to relate the chemical composition of the coffee grain to the beverage quality, including identification of possible molecular markers for flavor characterizing. This study was aimed at evaluating the changes in the proteomic profile of pulped and natural C. arabica grains dried in a yard or dryer at 60 °C. It was observed that fruits dried in a dryer at 60 °C showed an altered proteomic profile, with a reduction in the most abundant proteins compared to those yard-dried grains. Among the identified proteins, those involved in the metabolism of sugars and stress response were highlighted. Results have shown that post-harvest processes that impact coffee quality are related to changes in protein abundance, indicating that proteomic analysis may be effective in the identification of biochemical changes in coffee grains subjected to different post-harvest processes. [ABSTRACT FROM AUTHOR]

Abamectin is a microbial-derived pesticide widely used for control of agricultural pests. However, sustained use of abamectin has led to the development of resistance in some target species. Previous studies on arthropod resistance to abamectin have mainly used traditional biochemical and molecular approaches. To understand the responses of citrus red mite, Panonychus citri , exposed to abamectin, comparative proteomic analysis was conducted using two-dimensional electrophoresis (2-DE). A total of 26 distinct protein spots were present in response to abamectin exposure. Tandem mass spectrometry (MS/MS) identified 16 proteins that were mainly involved in energy metabolism and detoxification. Some remaining proteins were not identifiable, suggesting that they may be novel. The expression levels of transcripts associated with proteins were analyzed by quantitative reverse transcription PCR (qRT-PCR). Furthermore, to validate the proteomic data obtained in the present study, Western-blot experiment was performed and the expression of sHsp and PcE1 proteins were confirmed, respectively. Biological significance The citrus red mite has developed resistance to many acaricides, including abamectin. In the current study, we used the proteomic approaches involving 2-DE, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), and MS/MS to document changes in adult P . citri during 24 h of abamectin exposure. Abamectin stress induced a total of 16 differentially regulated proteins. The proteomic results were validated in mRNA expression patterns using qRT-PCR. This is the first analysis of differentially expressed proteins in P . citri exposed to abamectin. The results help clarify the physiological mechanisms of P . citri responses to abamectin exposure. [ABSTRACT FROM AUTHOR]

Certain clinical isolates of Tritrichomonas foetus infect the urogenital tract of cattle while others infect the gastrointestinal tract of cats. Previous studies have identified subtle genetic differences between these isolates with the term “genotype” adopted to reflect host origin. The aim of this work was to seek evidence of host-specific adaptation and to clarify the relationship between T. foetus genotypes. To do this we characterised the proteomes of both genotypes using two-dimensional gel electrophoresis (2DE) coupled with LC-MS/MS. Our comparative analysis of the data revealed that both genotypes exhibited largely similar proteoform profiles; however differentiation was possible with 24 spots identified as having a four-fold or greater change. Deeper analysis using 2DE zymography and protease-specific fluorogenic substrates revealed marked differences in cysteine protease (CP) expression profiles between the two genotypes. These variances in CP activities could also account for the pathogenic and histopathological differences previously observed between T. foetus genotypes in cross-infection studies. Our findings highlight the importance of CPs as major determinants of parasite virulence and provide a foundation for future host-parasite interaction studies, with direct implications for the development of vaccines or drugs targeting T. foetus . [ABSTRACT FROM AUTHOR]

Amoebic gill disease (AGD), caused by the marine ectoparasite Neoparamoeba perurans , is one of the most significant infectious diseases for Atlantic salmon ( Salmo salar ) aquaculture. Upon colonisation of the host gills, the parasite induces a marked hyperplasia and fusion of the lamellar epithelium, which can severely compromise the host if left untreated. In order to investigate the host response during the disease at a protein level, sequential gill samples were collected and analysed using two-dimensional electrophoresis (2DE), with peptides of interest subjected to LC-MS/MS. Samples were obtained from an experimental challenge using naïve Atlantic salmon smolts and cultured N. perurans . Sampling points were set at 1, 2, 3, 7, 14 and 21 days post-infection, and included both sub-clinical and clinical stages of the disease. A total of 23 proteins differentially expressed between non-infected and infected individuals were successfully identified by LC-MS/MS. Findings included upregulation of prohibitin, cyclophilin A, apolipoprotein A 1, ictacalcin, RhoGDP dissociation inhibitor α, components of the heat shock proteins 70 family and histones H3a and H4, and downregulation of peroxiredoxin-5 and cofilin. Among the most significant protein functions identified were cell cycle regulation, cytoskeletal regulation, oxidative metabolism and immunity. This is the first sequential proteomic analysis of gills during N. perurans infection. It is believed that the use of non-target screening techniques can contribute to the knowledge of gill responses to injury and pathogenic insults, including AGD. [ABSTRACT FROM AUTHOR]

Proteins play crucial roles in biological systems, thus studies comparing the protein pattern present in a healthy sample with an affected sample have been widely used for disease biomarker discovery. Although proteins containing metal ions constitute only a small proportion of the proteome, they are essential in a multitude of structural and functional processes. The correct association between metal ions and proteins is essential because this binding can significantly interfere with normal protein function. Employment of a metalloproteomic study of liver samples from diabetic rats permitted determination of the differential abundance of copper-, selenium-, zinc- and magnesium-associated proteins between diabetic, diabetic treatment with insulin and non-diabetic rats. Proteins were detected by ESI–MS/MS. Seventy-five different proteins were found with alterations in the metal ions of interest. The most prominent pathways affected under the diabetic model included: amino-acid metabolism and its derivates, glycogen storage, metabolism of carbohydrates, redox systems and glucose metabolism. Overall, the current methods employed yielded a greater understanding of metal binding and how type 1 diabetes and insulin treatment can modify some metal bonds in proteins, and therefore affect their mechanism of action and function. [ABSTRACT FROM AUTHOR]

Prostate cancer is the second most frequently diagnosed cancer in men worldwide. Currently prostate specific antigen (PSA) serum concentration is the most used prostate cancer marker, but it only shows limited specificity. Because PSA glycosylation is altered by prostate cancer, detecting glycosylation changes could increase PSA specificity as a prostate cancer marker. Changes in PSA glycosylation can modify its electrophoretic- behavior and techniques such as capillary zone electrophoresis (CZE) and two-dimensional electrophoresis (2-DE) could be applied to detect changes in PSA glycosylation. Most serum PSA is complexed with alpha-1 antichymotrypsin (ACT). To have access to most of the PSA, the complexed PSA has to be released as free PSA (fPSA); in addition, this total fPSA must be purified from the serum matrix so that it can be analyzed using CZE. In this work a methodology for isolating PSA from serum for its CZE analysis was established. By using PSA standard, the effect of this methodology, which combines conditions for dissociating complexed PSA and immunoaffinity chromatographic purification, was studied. It was seen that this highly repeatable sample treatment did not noticeably alter the circular dichroism (CD) spectrum or the CZE pattern of PSA standard. Therefore, as a proof-of-concept, the developed sample treatment was applied to serum from a cancer patient with a high PSA content. The following observations can be made from these experiments: first of all, the 2-DE pattern of serum PSA remained unchanged after sample treatment; second, as hypothesized, the established sample preparation methodology made it possible to obtain the CZE pattern of PSA from serum; and third, the CZE pattern of serum PSA and of PSA standard from seminal plasma of healthy individuals, both submitted to the sample treatment method, showed some differences regarding the proportion of CZE peaks of the glycoprotein. These differences could be related to possible changes in the linkages of peptide backbone, in glycosylation or in other post-translational modifications between samples from both origins. [ABSTRACT FROM AUTHOR]

This allergic reaction to snake venom was described to occur in patients after recurrent exposure through bites in amateur and professional snake handlers, which might be underestimated and contribute to fatal snakebites in victim, independently from the toxicity of the venom itself. Few allergens were identified from snake venoms by normal SDS-PAGE, which cannot separate the snake venom completely. In the present study, we identified nine potential allergens by two-dimensional (2D) electrophoresis followed by immunoblotting (named as allergenomics) in Protobothrops mucrosquamatus venom. By multidimensional liquid chromatography-ion trap mass spectrometry (MDLC-ESI-LTQ-MS/MS) analysis, six allergens showed sequence similarity to snake venom serine proteinases. Other allergens showed sequence similarity to snake venom metalloproteinase. These allergic reactions to snake venom allergens might contribute to fatal snakebites in victim, independently. [ABSTRACT FROM AUTHOR]

Around 15% of couples of childbearing age suffer from infertility; in 50% of these cases, the male factor is present. In this study, we investigated the association between anti-ODF2 autoantibody existence and the DNA fragmentation and apoptosis of sperm in oligozoospermia men. 35 fertile men and 57 oligozoospermia men are enrolled in this study as control and case groups, respectively. After the identification of ODF2 as a possible target of anti-sperm antibodies in sera of oligozoospermia men using two-dimensional gel electrophoresis followed by western blotting and mass spectrometry, the case group serums were screened for anti-ODF2 autoantibodies and divided into anti-ODF2 negative (N = 24) and positive (N = 33) subgroups to follow assays. The mRNA expression levels of ODF2, Caspases 3, 8, 9, BAX , and BCL-2 were evaluated via qRT-PCR in spermatozoa samples of mentioned groups. DNA fragmentation and apoptosis rate of spermatozoa in studied groups were assessed using an SDF kit and flow cytometry, respectively. Mass spectrometry showed that ODF2 is one of the anti-sperm antibodies targeted in oligozoospermia patients. 33 of 57 oligozoospermia men had anti-ODF2 autoantibody in their sera. An elevated expression of ODF2 mRNA was observed in spermatozoa of anti-ODF2+ patients compared to anti-ODF2- patients and controls. There was an increased expression level of Caspase 3, 8, 9 , and BAX and decreased expression of BCL-2 in spermatozoa of anti-ODF2+ patients compared to anti-ODF2- patients and controls. Noticeable increases in DNA fragmentation and apoptosis rate of anti-ODF2+ patients' spermatozoa were observed compared to anti-ODF2- patients and healthy controls spermatozoa. A positive correlation was observed between ODF-2 expression and DNF fragmentation and apoptosis rate of anti-ODF2+ patients' spermatozoa. Our results revealed that ODF2 is one of the main spermatozoa structural proteins, which is one of the anti-sperm antibodies targets, and its dysregulated expression may result in an increased rate of sperm DNA fragmentation and apoptosis. [ABSTRACT FROM AUTHOR]

By first time, 2DE protein profile of European eel seminal plasma has been determined. 14 different proteins corresponding to 9 major families were identified in seminal plasma, through hormonal treatment. Some of them play a part in sperm maturation, including carbonic anhydrase which is responsible for modulating the pH of seminal plasma, and warm temperature acclimation protein, which may play an important role in the final maturation of this species, due to the warm temperature of their spawning ground (in the Sargasso Sea). Sperm samples were classified into three motility categories depending on the percentage of motile cells, I: 0–25%, II: 25–50% and III: > 50%. Different protein profiles were observed depending on the sperm motility categories, specifically, with the apolipoproteins and complement C3. Higher numbers of proteins from the apolipoprotein family were registered at lower motilities; whereas the complement C3-like family was higher in the samples with the highest percentage of motile cells. These results suggest that the proteins linked to the transportation of lipids (apolipoprotein) and to the immune system (complement C3) may carry out their functions at different stages of spermatogenesis. Using SDS-PAGE analysis, 13 bands were identified, most of which migrated between 20 to 60 kDa. In the last weeks of treatment significant increases were observed in the percentage of motile spermatozoa, curvilinear velocity and beat cross frequency. This improvement in sperm quality coincided with a higher amount of proteins located at 19 KDa, therefore, this protein could be involved in sperm motility of the European eel. [ABSTRACT FROM AUTHOR]