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Pregnancy has its own protein dynamics, reflecting the hormonal profile. Quantitative and qualitative changes in plasma protein profile may provide useful information about this condition. Any alterations may be a signal heralding clinical or subclinical pathology. The objective of our study was to compare the plasma protein profile between selected months of pregnancy in cows for a better understanding gestation course. For this purpose, we collected blood from healthy pregnant (n = 30; n = 6 for each pregnancy stage) and non‐pregnant (C; n = 6) Holstein‐Friesian cows during a routine veterinary examination. Collected samples were selected according to pregnancy month (first, second, third, sixth, and ninth), prepared, and separated by two‐dimensional electrophoresis. The Delta‐2D program compared and statistically evaluated scanned gel images from the appropriate months. The mean volume of the spots was considered. The MALDI TOF/TOF spectrometer was used to identify statistically significant proteins. There were 11 distinct proteins found, including peptidyl‐prolyl cis‐trans isomerase F, oligoribonuclease, and PRELI domain‐containing protein 3B (all of them have the lowest abundance in the C group), alpha‐1B‐glycoprotein, L‐gulonolactone oxidase, hemopexin (first month with higher abundance than control), alpha‐2‐HS‐glycoprotein (significantly higher abundance in the first month than in remaining groups), ermin (absent in the first month and lower abundance in the third and sixth months than in the remaining groups and control), endophilin‐A2 (significant differences between the control and the second, third, sixth, and ninth months), apolipoprotein A‐I (significant difference between control and the first and sixth months), alpha‐1‐antiproteinase (significant difference between control and the ninth month). The study demonstrated the distinctions between plasma protein composition and alterations during the pregnancy course which may potentially serve as diagnostic tools. [ABSTRACT FROM AUTHOR]

Two‐dimensional electrophoresis (2DE) is a gel‐based protein separation method based on size and charge which is commonly used for the characterization of host cell proteins (HCPs) during drug development in biotech and pharmaceutical companies. HCPs are a heterogenous mixture of proteins produced by host cells during a biologics drug manufacturing process. Different gel electrophoresis methods including traditional 2D SDS‐PAGE with silver and SYPRO Ruby fluorescent dye staining as well as two‐dimensional difference gel electrophoresis (2D‐DIGE) were compared for their relative abilities to characterize HCPs. SYPRO Ruby was shown to be more sensitive than silver stain in the traditional 2D gels both with and without product protein present. Silver stain also displayed a significant preference for staining acidic proteins over basic ones while SYPRO Ruby was more consistent in imaging proteins across different isoelectric points. The non‐traditional method of 2D‐DIGE provides high resolution and reproducibility when comparing samples with similar protein profiles but was limited in imaging HCP spots due to its narrow dynamic range. Overall, 2DE is a powerful tool to separate and characterize HCPs and is optimized by choosing the best stain or method for each specific application. Using a combination of two or more different 2DE staining methods, when possible, provides the most comprehensive coverage to support the characterization of a complex mixture like HCPs. However, in instances where only one staining method can be used, SYPRO Ruby is shown to be the more reliable, more sensitive, and easier to use traditional staining method for most HCP‐based applications. [ABSTRACT FROM AUTHOR]

Boron, in the form of potassium tetraborate, has previously been found to be effective at inhibiting mango black spot disease, caused by Alternaria alternata. However, the mechanisms involved in this inhibition are largely unknown. In this study, A. alternata was treated in vitro with potassium tetraborate at a concentration of 5–10 mM for 48 or 72 h. The intracellular and mitochondrial proteins were extracted from mycelium and separated using two‐dimensional electrophoresis (2‐DE). Differentially expressed proteins (DEPs) were identified using bioinformatics tools and differences between protein spots were derived from mass spectrometry (MS). Using matrix‐assisted laser desorption ionization time‐of‐flight tandem mass spectrometry (MALDI‐ToF‐MS/MS), 96 intracellular and 56 mitochondrial DEPs were identified. The intracellular proteins identified were found to be involved in posttranslational modifications, protein turnover and chaperones, while the mitochondrial proteins were involved in electron transport chains. Our results demonstrate that various metabolic pathways are involved in the antifungal activity of boron. The differential expression of 20 genes was also verified at the mRNA level by reverse transcription‐quantitative PCR. Our study suggests that borate could be used as a potential substitute for synthetic fungicides to control this postharvest disease of mango fruits. [ABSTRACT FROM AUTHOR]

Moniliasis caused by the fungus Moniliophthora roreri is one of the most destructive cacao diseases in tropical America. In 2021, the presence of the pathogen was confirmed in cacao plantations in Brazil. This is the first proteomic study of the M. roreri protein profile of ungerminated spores and mycelium compared by 2D SDS‐PAGE (Two‐dimensional gel electrophoresis) associated with mass spectrometry. A total of 446 spots were detected on ungerminated spores gels and 402 spots on M. roreri mycelial gels. A total of 29 proteins were identified from the ungerminated spores, 21 of them were exclusive, and 53 were identified in the mycelium, 16 of them were exclusive. Most of the identified proteins at both development stages were categorized as being involved in metabolic processes, reduction/oxidation processes, and protein synthesis and folding. The interaction networks observed expand the known interactions of these proteins, resulting in eight functional clusters for proteins identified in ungerminated spores and seven clusters for mycelial proteins. In addition, a biological model of ungerminated spores and mycelium with cell location of proteins was constructed. The results contribute to a better understanding of the metabolic mechanisms of M. roreri during dormancy and vegetative development, which can support future studies for strategies to control moniliasis. [ABSTRACT FROM AUTHOR]

Common bean (Phaseolus vulgaris L.) seeds are an economical protein source rich in bioactive compounds with antioxidant activity. In this work, we evaluated processed seeds from two common bean cultivars regarding protein profile and antioxidant capacity before and after enzymatic digestion in vitro. We provided protein maps by two‐dimensional electrophoresis (2‐DE) of seeds germinated during 36 h and 72 h. The principal component analysis (PCA) showed that protein abundance had more variation on germination times than the cultivar ones. Seeds germinated for 36 h showed greater antioxidant capacity compared to germination for 72 h and cooking, before and after enzymatic digestion. In protein isolates, cooking associated with digestion provided better antioxidant capacity. Germination time influences protein expression as well as the antioxidant capacity of common beans. Furthermore, in vitro digestion increases the antioxidant capacity of cooked bean protein isolates. [ABSTRACT FROM AUTHOR]

Aim: To introduce a quantitative determination of heparan sulfate and dermatan sulfate by mass spectrometry and to compare it with two‐dimensional electrophoresis of the glycosaminoglycans in the amniotic fluid for the prenatal diagnosis of mucopolysaccharidoses type II (MPS II). Methods: Thirty pregnancies each with single fetus were subjected to amniocentesis at 16 weeks: 10 with a previously affected MPS II infant and 20 as controls. Prenatal diagnosis was done by both mass spectrometry two two‐dimensional electrophoresis. Results: Two‐dimensional electrophoresis showed four affected with MPS II and six unaffected fetuses. Mass spectrometry verified these results. Conclusion: Two‐dimensional electrophoresis of the glycosaminoglycans in amniotic fluid is a good qualitative method and mass spectrometry is a new accurate quantitative method for prenatal diagnosis of MPS II. Quantitative determination of glycosaminoglycans in amniotic fluid by mass spectrometry is both rapid and accurate. Prenatal diagnosis is recommended for at risk pregnancies and mass spectrometry offers speed and quantitation. [ABSTRACT FROM AUTHOR]

Comparative proteomic profiling of human follicular fluid (HFF) from polycystic ovary syndrome (PCOS) and non-PCOS patients who displayed low levels of fertility was carried out via two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry. HFF, an important reproductive fluid, was used for the proteomic analysis of PCOS patients to determine the effect of PCOS on folliculogenesis. HFF was obtained from 10 women (average age, 35 years) undergoing in vitro fertilization at two hospitals. The proteins of HFF were separated using 2-DE analysis and validated by Western blot assay. Approximately 250 protein spots were separated on the 2-DE gel. Among them, the expression levels of seven proteins were found to change at least 1.5-fold in the PCOS patient group. Three protein spots, albumin, uncharacterized protein 1, and uncharacterized protein 2, were downregulated in PCOS patients. However, four protein spots, gelsolin, vitamin D binding protein, serum albumin, and complement factor B, were upregulated in PCOS patient group. These proteins may serve as a panel of potential pathological biomarkers during fertilization and oocyte development. [ABSTRACT FROM AUTHOR]

Stress is one of the leading causes of male infertility, but its exact function in testosterone synthesis has scarcely been reported. We found that adult male rats show a decrease in bodyweight, genital index and serum testosterone level after continual chronic stress for 21 days. Two‐dimensional gel electrophoresis (2‐DE) and MALDI‐TOF‐MS analysis identified 10 differentially expressed proteins in stressed rats compared with controls. A strong protein interaction network was found to be centred on Atp5a1 among these proteins. Atp5a1 expression significantly decreased in Leydig cells after chronic stress. Transfection of Atp5a1 siRNAs decreased StAR, CYP11A1, and 17β‐HSD expression by damaging the structure of mitochondria in TM3 cells. This study confirmed that chronic stress plays an important role in testosterone synthesis by regulating Atp5a1 expression in Leydig cells. [ABSTRACT FROM AUTHOR]

Background: In the scientific literature, there are only a few manuscripts available on small animal faecal proteomics. Methods: In the present pilot study, this evaluation was performed using pooled faecal samples from 10 clinically healthy dogs and, for the first time, in 10 clinically healthy cats by mean of two‐dimensional electrophoresis followed by liquid chromatography‐tandem mass spectrometry. Results: Our results showed the presence of nine (albumin, alkaline phosphatase, chymotrypsin‐C‐like, cytosol aminopeptidase, elastase‐3B/proteinase E, immunoglobulins and nuclear pore membrane glycoprotein 210) and 14 (albumin, caspase recruitment domain‐containing protein, chymotrypsin‐like, deleted in malignant brain tumours 1 protein‐like, hypothetical protein LOC107375, immunoglobulin, kallikrein‐1, superoxide dismutase, transthyretin precursor, interstitial collagenase‐like) different proteins in canine and feline faeces, respectively. Conclusion: These preliminary findings document the presence of a range of proteins in the faeces of apparently healthy dogs and cats and may serve as a basis for larger, prospective studies to establish reference proteomic data against which diseased populations can be compared. [ABSTRACT FROM AUTHOR]

We aimed to identify proteins that were differentially regulated in spermatozoal samples collected from fertile healthy men (FHM) and infertile patients with varicocele (IFPV) before and after varicocelectomy. Seminal samples were collected from 20 IFPV before and after varicocelectomy and from 14 FHM as controls. Samples underwent seminal examination and proteomic analysis. Extracted spermatozoal proteins were analysed using two‐dimensional gel electrophoresis, and differentially regulated spermatozoal proteins (DRSPs) were identified. In particular, attention was placed on those DRSPs in which the concentration changed after varicocelectomy and corrected to approximate levels observed in FHM. Varicocelectomy significantly improved the sperm count and concentration in IFPV (p < 0.05). Proteomic analysis showed that 11 DRSPs were identified when comparisons were made among the three groups. Among these 11 proteins, change in the SERPIN A5 concentrations was notable because it was 100‐fold downregulated in pre‐operative IFPV samples and nearly resembled to control concentrations following varicocelectomy. Western blot analysis using an anti‐SERPIN antibody validated the changes observed in SERPIN A5 levels before and after varicocelectomy operation. Increase in SERPIN A5 after varicocelectomy may be due to improvement in semen quality, suggesting that SERPIN A5 is a potential seminal biomarker for assessment of semen quality in varicocele‐related infertility. [ABSTRACT FROM AUTHOR]

The rice cultivar BRS AG (Oryza sativa L.) is a cereal developed to serve as a raw material for ethanol production or animal feed, contributing to an increasing energy demand and the economic sustainability of the rice sector. However, only few studies have reported this new cultivar. As proteomic analysis is crucial in understanding the physiological and adaptive behavior of BRS AG rice leaves under biotic and abiotic stresses, the present study compared the following three precipitation protocols for the protein extraction of leaf tissue in two‐dimensional electrophoresis analysis: phenol method, trichloroacetic acid‐acetone method, and a third method developed in our laboratory (BIOPRO). The efficiency and repeatability of the protein extraction protocols were evaluated by two‐dimensional electrophoresis analysis. Our results indicate that the BIOPRO protocol was the most efficient in protein extraction as compared to the efficiency of other protocols. We detected a greater amount of protein (15.44 μg/μL) and 414 spots, and the BIOPRO protocol provided the best results regarding reproducibility, focusing, resolved spots, the intensity of spots, and single spot detection. This novel protocol is useful for extracting high‐quality proteins from leaves and presents an efficient removal of contaminants, which is directly linked to a better resolution for two‐dimensional electrophoresis analysis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Prepared chicken breast deterioration is a complex biochemical process, of which protein change is one of the main features. The present research focuses on the analysis of proteins related to the deterioration in quality of prepared chicken breast through differential proteomics analysis. RESULTS: The physicochemical indexes of prepared chicken breast showed that quality gradually decreased at the second week of refrigerated storage, while the deterioration of chicken breast meat was obvious at the third week. Three key time points of quality change were determined to be at 0th, 2th and 5th week, respectively. In addition, 39 differential proteins were successfully identified using two‐dimensional gel electrophoresis and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Most of the identified proteins showed significant differences in expression at the three key points of storage, of which actin, myosin, α‐1,4‐glucan phosphorylase, phosphoglucomutase 1, heat shock protein β‐1, tubulin β‐7 chain and skeletal muscle type tropomodulin (fragment) were closely related to the quality deterioration of prepared chicken breast, and thus potential indicator proteins to evaluate the quality of chicken breast. CONCLUSION: The current study indicated that the physicochemical quality of prepared breast notably changed during refrigerated storage. Three key time points of quality change in the storage process of prepared chicken breast were determined. Furthermore, differential proteomics identified the key proteins related to freshness, which provides a theoretical basis for exploring the mechanism of chicken breast deterioration. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Since Charles Darwin discovered growth movements of the shoot tip (coleoptile) toward light, the grass coleoptiles have long been used as a model system to study plant growth. Rapid growth of the coleoptile is vital for successful seed germination and early seedling establishment. However, the proteome changes underlying the rapid growth of the coleoptiles are not yet clear in the model plant maize (Zea mays). In the present study, we investigated proteome changes in vivo occurring in the rapidly growing coleoptiles of maize with two‐dimensional gel electrophoresis combined with mass spectrometry. A quantitative comparison of the proteomes at 1.5, 3, and 5 days after germination showed significant changes in protein profiles with coleoptile growth. As a result, 31 differential abundance proteins (DAPs) representing 44 protein spots were identified, of which 21 DAPs with increased abundances were implied in growth‐related processes, including translational initiation, transcription regulation, protein and other compound synthesis, H+‐transmembrane transport and cytoskeleton organization. The selected DAPs were confirmed by reverse transcription quantitative PCR and immunoblot analysis. We suggested that the rapid growth of the coleoptile is largely due to its ability to quickly enhance relevant cellular processes, especially the increased synthesis of the growth‐related DAPs. The content of indole‐3‐acetic acid, salicylic acid, and jasmonic acid decreased significantly, and the content of gibberellins first decreased and then increased during the elongation of coleoptile. This study provides new insight into the significance of proteome changes in coleoptile growth. [ABSTRACT FROM AUTHOR]

Brassinosteroids (BRs) represent a unique class of steroidal plant hormones that display pronounced growth‐promoting activity at very low concentrations. Although many efforts have been made to characterize the molecular basis of BR action, little is known about the mechanisms behind the growth‐promoting effect of BRs at protein level.Proteomic analysis of response to the steroid plant hormone 24‐epibrassinolide (EBR) in wheat seedling shoots (Triticum aestivum L.) was performed using two‐dimensional electrophoresis (2‐DE) and immunoblotting with highly specific antibodies (PY20) to phosphotyrosine. EBR‐modulated proteins and phosphotyrosine polypeptides were identified using MALDI‐TOF mass spectrometry.The study revealed that EBR‐stimulated growth of wheat seedlings was accompanied by changes in the content of multiple proteins as well as in tyrosine phosphorylation of numerous polypeptides. Among them, 22 differentially accumulated proteins and 13 phosphotyrosine proteins were identified. Based on their performed functions, the identified proteins are involved in physiological processes (photosynthesis, growth, energy and amino acid metabolism) closely associated with intensification of plant metabolism.The EBR‐induced changes in protein abundance and tyrosine phosphorylation profile may contribute to growth stimulation of wheat seedlings under the action of EBR. The obtained data suggest an important role for EBR in the activation of protein metabolism underlying fundamental physiological processes, including growth promotion. [ABSTRACT FROM AUTHOR]

Knowledge of the physiological and molecular mechanisms of drought responses is fundamental for developing genetically drought tolerant and high yielding crops. To understand molecular mechanism of drought tolerance of soybean (Glycine max L.), we compared leaf proteome patterns of in two genotypes GN‐3074 (drought tolerant) and GN‐2032 (drought‐sensitive) under drought stress during vegetative stage. Proteins were extracted from leaves of well‐watered and drought‐treated plants by using the trichloroacetic acid (TCA)–acetone precipitation method and analyzed by two‐dimensional polyacrylamide gel electrophoresis. Out 488 reproducibly detected and analyzed on two‐dimensional electrophoresis gels, 26 proteins showed significant changes in at least one genotype. The identification of 20 differentially expressed proteins using mass spectrometry revealed a coordinated expression of proteins involved in cellular metabolisms including photosynthesis, oxidative stress defense, respiration, metabolism process, signal transduction, phosphorus transduction, and methyl transduction which enable plant to cope with drought conditions. The most identified proteins involved in photosynthesis and oxidative stress defense system. The up‐regulation of several photosynthetic proteins and also high abundance of oxidative stress defense proteins in GN‐3074 genotypes as compare to GN‐2032 genotypes might reflect the fact that drought tolerance of GN‐3074 is due to effective photosynthetic machinery and more defense against oxidative stress. Our results suggest that soybean plant might response to drought stress by applying efficiently stay‐green mechanism through coordinated gene expression during vegetative stage. [ABSTRACT FROM AUTHOR]

Male accessory gland (MAG) secretory proteins affect female reproductive physiology and behaviour when they are delivered to the female at the time of mating along with sperm. In our study, proteomic approaches were used to identify MAG proteins of Leucinodes orbonalis, a monophagous and destructive pest of brinjal. A set of 117 and 186 MAG spots were observed in virgin and mated males, respectively, using two‐dimensional gel electrophoresis (2D‐GE). Among the identified spots, 49 are unique to the virgin, whereas 118 are unique to mated MAGs, with 68 spots found to be common. The differentially expressed MAG proteins, 14 are up‐regulated, and 16 are down‐regulated in virgin after mating. We used matrix‐assisted laser desorption ionization (MALDI)–mass spectrometry (MS) to identify the 13 unique proteins in the MAG of virgin L. orbonalis and analysed the data of the Swiss‐Prot database using the Mascot search engine. The proteins were identified as proteolysis regulators, lipid transporters, olfactory proteins, metabolism‐related proteins, DNA‐binding proteins, and hexamerins. This is the first report on the gel‐based protein analysis of L. orbonalis MAGs. [ABSTRACT FROM AUTHOR]

Talazoparib (TAL) has been effectively used for the treatment of gBRCA1/2‐mutated HER2‐negative metastatic breast cancer. However, acquired resistance to TAL remains a major challenge that impedes the clinical success of TAL treatment. Therefore, elucidation of proteins and pathways that contribute to or are affected by the TAL resistance is urgently needed to improve the treatment response and provide novel treatment strategies for advanced metastatic breast cancers. Herein, we aimed to investigate the altered protein signatures in TAL‐resistant triple‐negative breast cancer (TNBC) cells by comparing with the TNBC parental cell line via proteomic analysis. After validation of TAL‐resistance by WST‐1 and Annexin V analysis, two‐dimensional gel electrophoresis (2DE)‐based proteomic analysis coupled to matrix‐assisted laser desorption/ionization (MALDI)–time of flight (TOF) mass spectrometry was performed to identify differentially regulated proteins. The findings revealed the identities of 10 differentially regulated proteins in TAL‐resistant TNBC cells whose bioinformatic analysis predicted changes in EGF/FGF signaling pathways as well as in the AMPK signaling pathway. In addition, phosphorylation/dephosphorylation dynamics were predicted to be altered in TAL‐resistant cells. The proteins identified in this study might be the targets to overcome TAL resistance for the treatment of TNBC. [ABSTRACT FROM AUTHOR]

Canine mammary tumours (CMTs) are regarded as invasive with a high rate of recurrent and metastasis in intact female dogs. Tumour diagnosis, therefore, is an important step in predicting and monitoring tumour progression. This study was designed to identify protein expression on CMTs by employing a proteomic approach. The primary cell culture from benign mixed tumour, simple carcinoma, complex carcinoma and normal mammary gland were established, and two‐dimensional electrophoresis (2DE) was subsequently performed. The different spots on each sample type were collected for identification using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The results indicated that cytokeratin 5 (CK5) and transketolase (TKT) were identified in benign mixed tumour cells and complex carcinoma cells. In contrast, cytokeratin 18 (CK18) and pyruvate kinase PKM were identified in simple carcinoma cells. Moreover, alpha‐2‐HS‐glycoprotein tumour antigen was identified specifically in complex carcinoma cells. In addition, ATP‐dependent 6‐phosphofructokinase platelet type and elongation factor 2 proteins were observed in benign cells. In conclusion, all expressed proteins in this study have been recognized for acting as their expression that differs from healthy mammary epithelial cells. Expectantly, this study identified the expressed proteins that might be useful in further diagnostic biomarker studies on CMTs. [ABSTRACT FROM AUTHOR]

Introduction: 15‐deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2) causes neuronal apoptosis independently of its nuclear receptor, peroxysome‐proliferator activated receptor γ. Its membrane receptor, chemoattractant receptor‐homologous molecule expressed on Th2 cells (CRTH2), did not also mediate the neurotoxicity of 15d‐PGJ2. In the present study, we ascertained whether membrane targets beside CRTH2 were involved in the neurotoxicity of 15d‐PGJ2. Methods: Neuronal membrane targets for 15d‐PGJ2 were separated by two‐dimensional electrophoresis, identified by proteomic approach. Their localizations were detected by microscopic immunofluorescence study. Cell viability and apoptosis was evaluated by MTT‐reducing activity and caspase‐3 activity, respectively. Results: Voltage‐dependent anion channel 1 (VDAC1) was identified as one of membrane targets for 15d‐PGJ2. Modification of VDAC1 with 15d‐PGJ2 was detected by pull‐down assay. VDAC1 was detected in the plasma membrane and localized on the neuronal cell surface. VDAC1 was partially colocalized with membrane targets for 15d‐PGJ2. The anti‐VDAC antibody significantly attenuated the neurotoxicity of 15d‐PGJ2, accompanied by the suppression of the 15d‐PGJ2‐stimulated caspase‐3. Conclusion: These findings suggested that the plasmalemmal VDAC might be involved in the neurotoxicity of 15d‐PGJ2. [ABSTRACT FROM AUTHOR]

Abiotrophia defectiva is a species of nutritionally variant streptococci that is found in human saliva and dental plaques and that has been associated with infective endocarditis. In our previous study, it was found that A. defectiva could bind specifically to saliva‐coated hydroxyapatite beads (SHA). This study identified a cell surface component of A. defectiva that promotes adherence to SHA beads. The binding of A. defectiva to SHA was reduced in the presence of antibodies against human proline‐rich protein (PRP); these results suggested that PRP may be a critical component mediating interactions between A. defectiva and the salivary pellicle. Two‐dimensional gel electrophoresis of whole A. defectiva cells followed by Far‐Western blotting was conducted by probing with synthetic peptides analogous to the binding region of PRP known as PRP‐C. The results indicate that an A. defectiva protein of 37 kDa interacts with PRP‐C. The results of amino‐terminal sequencing of the adhesive A. defectiva protein revealed significant similarity to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Recombinant GAPDH bound to immobilized PRP‐C in a dose‐dependent manner and binding of A. defectiva to SHA or to PRP was reduced in the presence of anti‐GAPDH antiserum. Western blotting or electron immunomicroscopic observations with anti‐GAPDH antiserum revealed that this protein was expressed in both cytosolic and cell wall fractions. These results suggest that A. defectiva could specifically bind to PRP via interactions with cell surface GAPDH; the findings suggest a mechanism underlying A. defectiva‐mediated adherence to saliva‐coated tooth surfaces. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate improved protein extraction and two‐dimensional electrophoresis (2DE) separation methods with Japanese reference human hair (JRH); to determine whether fibre curvature is related to protein composition in curly and straight Japanese women's human hair (JHH) samples; and to identify proteins from JRH 2DE maps and expression differences between curly and straight JHH. Methods: Hair keratin and keratin‐associated proteins (KAPs) were extracted intact with dithiothreitol or tris(2‐carboxyethyl) phosphine from JRH or from curved or straight JHH. Extracted proteins were isoelectric‐focused on first‐dimensional pH gradient gel strips, then separated by molecular weight on laboratory‐made, second‐dimension, large format gels. The software compared protein abundance between duplicate 2DE gels of curved and straight JHH. Thirty‐eight proteins from a JRH 2DE gel were enzyme‐cleaved for MALDI‐TOF‐MS analysis to determine peptide composition, and where possible, de novo sequencing gave peptide sequence data. An in‐house human hair protein database incorporating ninety‐eight annotated protein sequences assisted MS analysis. Results: 2DE gels of tris(2‐carboxyethyl) phosphine‐extracted JRH improved keratin and KAP resolution and number compared to those of dithiothreitol‐extracted JRH and published commercially made second‐dimensional gels. Silver‐stained 2DE gels of the straight or curved JHH sets were remarkably similar. Over‐staining to reveal basic proteins caused poor resolution of the major acidic protein classes. Software comparisons of fifty‐nine resolved proteins revealed two were significantly different in abundance between curved and straight hairs but in insufficient amounts for MS analysis. MS identified twelve proteins from a JRH CBBG‐stained 2DE gel: six type II keratins, three type I keratins and three high sulphur proteins. A further eight were potential conformational isoforms and isoelectric variants of the identified proteins bringing the total to twenty identified or partially identified proteins. Conclusion: Root‐end human hair extraction with tris(2‐carboxyethyl) phosphine improves protein resolution and visualizes more proteins on large format 2DE gels. The two minor protein differences between duplicate straight or curved JHH 2DE gels were unlikely to change fibre structure from straight to curved hair. MS results confirmed that multiple isoforms exist of various hair proteins. Low sequence coverage prevented distinction between members in rows of homologous protein spots of similar molecular weight. [ABSTRACT FROM AUTHOR]

Potato tuberization is a complicated biological process regulated by multiple phytohormones, in particular cytokinins (CKs). The information available on the molecular mechanisms regulating tuber development by CKs remains largely unclear. Physiological results initially indicated that low 6‐benzylaminopurine (BAP) concentration (3 mg l−1) advanced the tuberization beginning time and promoted tuber formation. A comparative proteomics approach was applied to investigate the proteome change of tuber development by two‐dimensional gel electrophoresis in vitro, subjected to exogenous BAP treatments (0, 3, 6 and 13 mg l−1). Quantitative image analysis showed a total of 83 protein spots with significantly altered abundance (>2.5‐fold, P < 0.05), and 55 differentially abundant proteins were identified by MALDI‐TOF/TOF MS. Among these proteins, 22 proteins exhibited up‐regulation with the increase of exogenous BAP concentration, and 31 proteins were upregulated at 3 mg l−1 BAP whereas being downregulated at higher BAP concentrations. These proteins were involved in metabolism and bioenergy, storage, redox homeostasis, cell defense and rescue, transcription and translation, chaperones, signaling and transport. The favorable effects of low BAP concentrations on tuber development were found in various cellular processes, mainly including the stimulation of starch and storage protein accumulation, the enhancement of the glycolysis pathway and ATP synthesis, the cellular homeostasis maintenance, the activation of pathogen defense, the higher efficiency of transcription and translation, as well as the enhanced metabolite transport. However, higher BAP concentration, especially 13 mg l−1, showed disadvantageous effects. The proposed hypothetical model would explain the interaction of these proteins associated with CK‐induced tuber development in vitro. [ABSTRACT FROM AUTHOR]

Summary: A diet based on sunflower cake for lambs was assayed in order to reuse biodiesel industrial by‐products with the aim of reducing livestock costs and evaluating their influence on meat quality. To achieve these goals, sixteen male lambs were fed diets containing different levels of sunflower cake (control, 5%, 10% and 15%). Afterwards, their semimembranosus muscles were analysed by two‐dimensional electrophoresis coupled to mass spectrometry and their functional protein association was examined using STRING. Structural and metabolic proteins in the lambs' proteomes changed significantly according to their diet. Fifteen proteins showed significant changes caused by the inclusion of sunflower cake, and the most differentially abundant structural proteins were detected in 2‐DE gels from the lambs. Differentially abundant metabolic proteins such as ENO3 (enolase 3), MDH1 (malate dehydrogenase) and ALDH1A1 (retinal dehydrogenase) have been proposed as biomarkers of quality parameters in other species. [ABSTRACT FROM AUTHOR]

Vibrio alginolyticus is one of the most serious causative agents of diseases in cultured marine fish and shellfish. However, the characteristics of virulence factors in pathogenic V. alginolyticus are poorly known. To gain insight into fish diseases caused by V. alginolyticus, we carried out two‐dimensional gel electrophoresis (2‐DE) combined with MALDI‐TOF mass spectrometry to identify uniquely expressed proteins in the disease‐causing V. alginolyticus. V. alginolyticus strains were isolated from marine environments and diseased fish obtained from southern Thailand. We identified seven unique proteins in the disease‐causing V. alginolyticus strain. Among those, the outer membrane protein A (OmpA) had the strongest expression. Therefore, the function of this protein was further analysed. To investigate the role of OmpA protein, an in‐frame deletion mutant of ompA was constructed using the homologous recombination method. Although the ompA mutant V. alginolyticus strain (ΔompA) grew normally, the mutant exhibited a significant defect in the swarming ability and the biofilm formation. Furthermore, Galleria mellonella larvae injected with the mutant bacteria had a significantly greater survival percentage than those injected with the wild‐type strain, demonstrating that OmpA protein is required for the pathogenicity of V. alginolyticus. Together, this study suggests a potential target for vaccine development against pathogenic V. alginolyticus strain. [ABSTRACT FROM AUTHOR]

The aim of the study was to evaluate the influence of dietary supplementation with inulin extract from chicory root and dried chicory root on the protein profile of the renal cortex and medulla of growing pigs. The experiment was carried out on renal cortex and medulla tissue collected from 24 50‐day‐old PIC x Penarlan P76 crossbred piglets (males). Animals were divided into three dietary groups (n = 8) and fed with a control diet, diet supplemented with 2% inulin extract from chicory root and a diet supplemented with 4% dried chicory root. Kidney samples were collected after 40 days of feeding, and renal cortex and medulla proteins were separated by two‐dimensional electrophoresis. Protein identification was performed using MALDI‐TOF mass spectrometry. The diet supplemented with 2% chicory inulin induced significant expression changes of 20 and 26 protein spots in the renal cortex and medulla respectively. Supplementation with 4% dried chicory root triggered changes in the expression of 44 and 24 proteins in the renal cortex and medulla respectively. Both forms of chicory inulin‐type fructans effectively affected the expression of proteins involved in energy metabolism, heat shock proteins and other chaperones, cytoskeletal and cytoskeleton‐related proteins, as well as other proteins. Additionally, changes in transferrin abundance in both experimental groups suggested the significance of chicory fructan supplementation for iron absorption and bioavailability. In conclusion, 2% inulin extract from chicory root and 4% dried chicory root exerted a similar effect on changes in renal protein expression; however, more pronounced alterations were induced by dried chicory root. Nevertheless, further studies are needed for better understanding the mechanism underlying the effect of chicory inulin‐type fructans and their fermentation end products on the kidneys of growing pigs. [ABSTRACT FROM AUTHOR]

Mango anthracnose caused by the fungal pathogen Colletotrichum gloeosporioides results in substantial economic losses in postharvest fruits. Borate is an effective fungicide against C. gloeosporioides. It appears to induce mitochondrial degradation and dysfunction in the fungal conidia, but the underlying molecular mechanism remains unclear. This study investigated the effects of borate on this pathogen by examining its mitochondrial proteins, which were extracted from C. gloeosporioides mycelium after borate treatment (5 or 10 mm potassium tetraborate for 48 and 72 h) and characterized using proteomic analysis (two‐dimensional electrophoresis, 2‐DE; and mass spectrometry, MS) and bioinformatics. In total, 600 protein spots were separated by 2‐DE, and 115 of these appeared to be differentially expressed proteins (DEPs). Among the DEPs, 58 were identified using MALDI‐TOF‐TOF MS/MS. Borate treatment was found to alter the expression levels of proteins involved in seven different metabolic pathways, mostly related to energy supply and/or biosynthesis. The effect of borate on mRNA expression levels of 17 genes involved in these metabolic pathways was confirmed by real‐time quantitative reverse transcription PCR (qRT‐PCR). The results suggest that borate might have distinct effects on mitochondrial function in C. gloeosporioides and could be used to control anthracnose in postharvest mango fruits. [ABSTRACT FROM AUTHOR]

The present study was designed to investigate proteomic differences in duck breast muscle during the early postmortem storage period. The meat quality was evaluated at 0 hr and 24 hr postmortem at 4°C in Pekin ducks, black Muscovy ducks and Mule ducks. Differentially expressed proteins were detected by two‐dimensional gel electrophoresis (2‐DE) and matrix‐assisted laser desorption ionization‐time‐of‐flight mass spectrometry (MALDI‐TOF/TOF MS) at 0 hr and 24 hr postmortem in the three duck breeds. The results showed that 53 proteins spots were differentially expressed at 0 hr and 24 hr postmortem at 4°C in Pekin ducks, 75 spots in black Muscovy ducks, and 72 spots in Mule ducks. A total of 30 (10 spots for each breed) were selected for identification by mass spectrometry. Seven proteins were identified in Pekin ducks, eight in black Muscovy ducks and seven in Mule ducks. Moreover, the above results obtained by 2‐DE and MALDI‐TOF/TOF MS were confirmed by western blotting. To our knowledge, this study is the first to provide insights into the protein profiles of ducks during postmortem storage and provides a better understanding of the biochemical processes that contribute to duck meat quality. [ABSTRACT FROM AUTHOR]

Obstructive sleep apnea (OSA) affects an estimated 20% of adults worldwide and has been associated with electrical and structural abnormalities of the atria, although the molecular mechanisms are not well understood. Here, we used two‐dimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS) to investigate the proteins that are dysregulated in the atria from severe and moderate apnea when compared to control. We found enzymes involved in the glycolysis, beta‐oxidation, electron transport chain and Krebs cycle to be down‐regulated. The data suggested that the dysregulated proteins may play a role in atrial pathology developing via chronic obstructive apnea and hypoxia. Our results are consistent with our previous 1D‐PAGE and nanoLC‐MS/MS study (Channaveerappa et al, J Cell Mol Med. 2017), where we found that some aerobic and anaerobic glycolytic and Krebs cycle enzymes were down‐regulated, suggesting that apnea may be a result of paucity of oxygen and production of ATP and reducing equivalents (NADH). The 2D‐PAGE study not only complements our current study, but also advances our understanding of the OSA. The complete mass spectrometry data are available via ProteomeXchange with identifier PXD011181. [ABSTRACT FROM AUTHOR]

Differentially expressed aqueous soluble proteins between Mycobacterium tuberculosis H37Ra and H37Rv were identified. The protein extracts were separated by two‐dimensional gel electrophoresis followed by tandem mass spectrometric analysis. Twelve proteins were detected to be differentially expressed significantly between virulent strain H37Rv and attenuated strain H37Ra. The differentially expression of these proteins was validated by a recently isolated clinical virulent strains of M. tuberculosis, TB138. Out of the 12 proteins identified, which consisted of ten upregulated and two downregulated proteins, nine were belonged to intermediate metabolism and respiration protein group, two were in lipid metabolism, and one protein was involved in information pathways and virulence. Among these proteins, two of the upregulated proteins, namely, mmsA and pntAa, showed a consistent expression pattern in both virulent mycobacterium strains. These proteins can serve as potential biomarkers for the intervention treatment of TB. [ABSTRACT FROM AUTHOR]

Rationale: Influenza A viruses (IAVs) are still a threat to human health and life. The process of virus infection involves a series of biological regulations, such as signal transduction, that may be closely linked with the function of glycoproteins. However, the number and level of glycoproteins is low compared with other proteins in the whole protein pool. Methods: Viruses obtained from chicken embryos were purified by sucrose gradient centrifugation. PNGase F enzyme was then used to remove the glycan modification, followed by two‐dimensional electrophoresis (2DE) to separate the hemagglutinin1 (HA1) glycoprotein. In‐gel digestion was used to obtain peptides that were detected by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Results: Remarkably, we found five isoforms of HA1 with the same molecular weight but different isoelectric points. Furthermore, HA1 treatment with PNGase F enzyme changed all but one protein spot from 2DE, indicating that the different HA1 isoforms in 2DE were a result of different glycosylation modifications. Conclusions: The difference in isoelectric points of these HA1 isoforms was caused by glycan modification. This method provides a new approach for the study of glycosylation of the proteome for viruses or any other organisms. [ABSTRACT FROM AUTHOR]

Abstract: Here, we developed an integrated course based on two dimensional‐electrophoresis and spectrometry mass (2DE‐MS) technique for undergraduate students to help them learn proteomic techniques. The soluble proteins in wild type and gene knockout bacteria were separated by 2DE and the differently expressed proteins were identified by MS analysis. The proteomic data was finally confirmed by RT‐PCR detection. The separated experiments of 2DE, MS, RNA isolation, RT‐PCR, as well as essential bioinformatic analysis, were integrated into a one‐week course, which provided students an opportunity to systematically understand the proteomic techniques and their applications in current scientific research. © 2018 by The International Union of Biochemistry and Molecular Biology, 46:354–360, 2018. [ABSTRACT FROM AUTHOR]

Abstract: Nilaparvata lugens (stal) is a rice pest and contains long‐winged and short‐winged varieties, called the wing differentiation. This study compared the protein profiles of the two wing‐types in females and two wing‐disc types 5th‐instar females by two‐dimensional electrophoresis analysis. We detected 172 and 174 protein spots in adults and 5th‐instar nymphs, respectively. The number of proteins with higher content in the long‐winged (disc) individuals is much more than that in the short‐winged (disc) individuals. A total of 32 differential protein spots were found, of which 20 were successfully identified. Their main function is about catabolic process, fiber and nucleoside binding, and they constitute 52 protein–protein interactions, which is around the glycolysis as the core. These results enrich the research on the protein Level in wing development, and provide more references for future studies. [ABSTRACT FROM AUTHOR]

The identification of phytopathogen proteins that are differentially expressed during the course of the establishment of an infection is important to better understand the infection process. In vitro approaches, using plant extracts added to culture medium, have been used to identify such proteins, but the biological relevance of these findings for in planta infection are often uncertain until confirmed by in vivo studies. Here, we compared the proteins of Pectobacterium carotovorum ssp. carotovorum strain PccS1 differentially expressed in Luria-Bertani medium supplemented with extracts of the ornamental plant Zantedeschia elliotiana cultivar 'Black Magic' ( in vitro) and in plant tissues ( in vivo) by two-dimensional electrophoresis coupled with mass spectrometry. A total of 53 differentially expressed proteins (>1.5-fold) were identified (up-regulated or down-regulated in vitro, in vivo or both). Proteins that exhibited increased expression in vivo but not in vitro, or in both conditions, were identified, and deletions were made in a number of genes encoding these proteins, four of which ( clpP, mreB, flgK and eda) led to a loss of virulence on Z. elliotiana, although clpP and mreB were later also shown to be reduced in growth in rich and minimal media. Although clpP, flgK and mreB have previously been reported as playing a role in virulence in plants, this is the first report of such a role for eda, which encodes 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, a key enzyme in Entner-Doudoroff metabolism. The results highlight the value of undertaking in vivo as well as in vitro approaches for the identification of new bacterial virulence factors. [ABSTRACT FROM AUTHOR]

BACKGROUND Proteins enzymatic digestion is a very complex process, during which some components are degraded, whereas others remain in an unchanged form. Moreover, enzymatic hydrolysis is one of the most popular methods used to reduce the allergenicity of food proteins. In the present study, the efficiency of enzymatic hydrolysis of lupin seed proteins was assessed by proteomic analysis as performed by two-dimensional gel electrophoresis (2- DE) coupled with mass spectrometry identification. Two digestion systems were used: oriented digestion carried out by trypsin and model in vitro digestion mimicking the conditions present in the gastrointestinal tract. RESULTS The comparisons of 2- DE maps of proteins isolated form different lupin seed species revealed that the differences in proteins expression were observed mainly in the central parts of gels (i.e. in the molecular weight range from 20 to 70 kDa, and the pH range 5-7). In total, 27 differentially expressed proteins spots were successfully identified by mass spectrometry analysis. An important reduction in the number of proteins spots on 2- DE maps was observed when trypsin and the in vitro digestion model were applied. The protein spot insensitive to digestion in both hydrolysis systems was identified as β-conglutin. CONCLUSIONS The results of the present study provide insight into the nature of the digestion process that may take place after lupin seed protein intake and highlight the important fact that some of the proteins are insensitive to digestive enzyme activity. Moreover, evaluation of digestion activity of trypsin towards lupin seed proteins may be used for the development of specific processes with respect to hypoallergenic food production. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Currently, a wide array of plant preparations exerting health-promoting properties are commonly used as feed additives. Among them, Cichorium intybus L. have gained considerable attention as a source of compounds showing prebiotic character. Large body of evidence suggests that products of prebiotic fermentation (short-chain fatty acids) may influence the expression of genes encoding liver enzymes involved in the regulation of energetic metabolism. Given the above, the present study was aimed at estimating the influence of a diet supplemented with chicory root or water extract of chicory inulin on liver proteome in growing pigs. The study was performed on 24 castrated male piglets ( PIC × Penarlan P76). Animals were assigned to three equal groups ( n = 8) and fed cereal-based isoenergetic diets: control and supplemented with 2% of inulin extract from chicory root or 4% of dried chicory root. Liver proteins were separated using two-dimensional electrophoresis, followed by the identification of statistically valid protein spots with the aid of MALDI- TOF mass spectrometry. Both experimental factors significantly modulated the expression of liver proteins associated with energetic metabolism, particularly those involved in cholesterol and triglyceride metabolism. Additionally, both dietary additives induced increased expression of proteins involved in hepatocyte protection against oxidative stress. In the present study, we have shown for the first time that diet supplementation with dried chicory root or inulin caused significant changes in the expression of liver cytoskeletal proteins. Close attention should be paid to the downregulation of cytokeratin 18, hepatic acute phase protein that can enhance the anti-inflammatory properties of inulin-type fructans. [ABSTRACT FROM AUTHOR]

Host cell proteins (HCP) are a problematic set of impurities in downstream processing (DSP) as they behave most similarly to the target protein during separation. Approaching DSP with the knowledge of HCP separation behavior would be beneficial for the production of high purity recombinant biologics. Therefore, this work was aimed at characterizing the separation behavior of complex mixtures of HCP during a commonly used method: anion-exchange chromatography (AEX). An additional goal was to evaluate the performance of a statistical methodology, based on the characterization data, as a tool for predicting protein separation behavior. Aqueous two-phase partitioning followed by two-dimensional electrophoresis provided data on the three physicochemical properties most commonly exploited during DSP for each HCP: pI (isoelectric point), molecular weight, and surface hydrophobicity. The protein separation behaviors of two alternative expression host extracts (corn germ and E. coli) were characterized. A multivariate random forest (MVRF) statistical methodology was then applied to the database of characterized proteins creating a tool for predicting the AEX behavior of a mixture of proteins. The accuracy of the MVRF method was determined by calculating a root mean squared error value for each database. This measure never exceeded a value of 0.045 (fraction of protein populating each of the multiple separation fractions) for AEX. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1453-1463, 2016 [ABSTRACT FROM AUTHOR]

BACKGROUND Data analysis of omics data should be performed by multivariate analysis such as principal component analysis ( PCA). The way data are clustered in PCA is of major importance to develop some classification systems based on multivariate analysis, such as soft independent modeling of class analogy ( SIMCA). In a previous study a one-class classifier based on SIMCA was built using microarray data from a set of potatoes. The PCA grouped the transcriptomic data according to varieties. The present work aimed to use PCA to verify the clustering of the proteomic profiles for the same potato varieties. RESULTS Proteomic profiles of five potato varieties (Biogold, Fontane, Innovator, Lady Rosetta and Maris Piper) were evaluated by two-dimensional gel electrophoresis (2- DE) performed on two immobilized pH gradient ( IPG) strip lengths, 13 and 24 cm, both under pH range 4-7. For each strip length, two gels were prepared from each variety; in total there were ten gels per analysis. For 13 cm strips, 199-320 spots were detected per gel, and for 24 cm strips, 365-684 spots. CONCLUSION All four PCAs performed with these datasets presented clear grouping of samples according to the varieties. The data presented here showed that PCA was applicable for proteomic analysis of potato and was able to separate the samples by varieties. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Most biomaterials at present have sufficient mechanical properties; however compliance with standards for biocompatibility is often not sufficient in clinical practice. This may be due to the complexity of biological systems in general and the diversity of individual responses to these materials by implant recipients. Significant improvement of biocompatibility must involve surface modification of implants, which in the future will make it possible to introduce individually selected types of surface modification for individual recipients. The key to this technology seems to be understanding the processes occurring at the site of contact of the implant with the tissue. Processes resulting from the stress generated by the contact of the biomaterial surfaces were observed with endothelial cells line EA.hy926, and it was demonstrated that differently modified surfaces of medical steel (polished medical steel and medical steel coated with Parylene C and nanocrystalline diamond) cause diverse cellular response in cells grown on these surfaces, on both the cellular (cell morphology and cell survival) and molecular (transcriptome and proteome profiles) levels. The herein presented observations are a good starting point not only for further research and the development of far-reaching personalization of medical implants, but also to study the potential use of cells as a specific sensor capable of recognizing different surfaces with which these cells come into contact. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 880-893, 2016. [ABSTRACT FROM AUTHOR]

Protein extraction for two-dimensional electrophoresis from tissues of recalcitrant species is quite problematic and challenging due to the low protein content and high abundance of contaminants. Proteomics in Shorea robusta is scarcely conducted due to the lack of a suitable protein preparation procedure. To establish an effective protein extraction protocol suitable for two-dimensional electrophoresis in Shorea robusta, four procedures (borate buffer/trichloroacetic acid extraction, organic solvent/trichloroacetic acid precipitation, sucrose/Tris/phenol, and organic solvent/phenol/sodium dodecyl sulfate) were evaluated. Following these, proteins were isolated from mature leaves and were analyzed for proteomics, and also for potential contaminants, widely reported to hinder proteomics. The borate buffer/trichloroacetic acid extraction had the lowest protein yield and did not result in any banding even in one-dimensional electrophoresis. In contrast, organic solvent/phenol/sodium dodecyl sulfate extraction allowed the highest protein yield. Moreover, during proteomics, organic solvent/phenol/sodium dodecyl sulfate extracted protein resolved the maximum number (144) of spots. Further, when proteins were evaluated for contaminants, significant (77-95%) reductions in the nucleic acids, phenol, and sugars were discernible with refinement in extraction procedure. Accumulated data suggested that the organic solvent/phenol/sodium dodecyl sulfate extraction was the most effective protocol for protein isolation for proteomics of Shorea robusta and can be used for plants that have a similar set of contaminants. [ABSTRACT FROM AUTHOR]

This study was conducted to unravel the variation in meat quality between tender (psoas major, PM) and less tender (longissimus lumborum, LL) muscles of Indian water buffaloes ( B ubalus bubalis). PM and LL were subjected to physicochemical analysis, ultrastructural study and proteome characterization using two-dimensional gel electrophoresis (2- DE) and mass spectrometry. Higher ( P < 0.05) muscle fiber diameter and Warner-Bratzler shear force was observed in LL, whereas higher ( P < 0.05) water-holding capacity and myofibrillar protein extractability was observed in PM. Transmission electron microscopy revealed higher ( P < 0.05) sarcomere length in PM compared with LL. Proteome analysis using 2- DE revealed 123 differentially abundant proteins in PM and LL. The MALDI- TOF-TOF MS analysis of selected protein spots from LL and PM with significant ( P < 0.05) differences identified the proteins mainly consisting of calcium-transporting ATPase. Practical Applications Owing to its low fat, low cholesterol and other healthier attributes, meat produced from water buffaloes has gained increased popularity in several Asian and African countries. Tenderness, which is considered to be the most important attribute of meat quality and a trait highly valued by consumers, varies substantially between muscles. In spite of advancements in optimizing tenderness, an unacceptable level of variation does exist. Findings from the present study illustrate the basis of tenderness variability in psoas major and longissimus lumborum muscles of Indian water buffaloes. Our results indicated complex nature of tenderness development and demonstrated a significant variation in biochemical parameters, ultrastructure and proteome expression. Understanding the variation in texture between muscles is a logical first step to develop strategies for better utilization and for delivering highly palatable product to consumers. This study suggests the necessity to develop muscle-specific tenderization strategies to minimize the variation between tender and less tender muscles. [ABSTRACT FROM AUTHOR]

Nine tilapia Oreochromis niloticus group B streptococcus ( GBS) strains differing in serotype and genotype were selected and paired. Two-dimensional difference gel electrophoresis (2D DIGE) and matrix-assisted laser-desorption ionization time-of-flight-mass spectrometry ( MALDI-TOF-MS) were used to analyse the protein profiles of the strain pairs. Forty-three proteins corresponding to 66 spots were identified, of which 35 proteins were found in the seven selected strain pairs that represented pairs differing in genotype and serotype. Among the 35 proteins, numbers of differentially expressed proteins in strains of different serotypes were greater than found in strains of different genotypes, suggesting that serotype plays a more essential role than genotype in the differential protein expression among GBS strains. No distinct pattern was found with respect to genotype and the protein expression profile of GBS strains. Several proteins were identified as surface-associated cytoplasmic proteins that possessed the typical immunity-eliciting characteristics of surface proteins. The identified proteins were found to be involved in 16 biological processes and seven Kyoto encyclopaedia of genes and genomes (KEGG) pathways. The data, for the first time, identified differentially expressed proteins in O. niloticus GBS strains of different serotypes, which play a major role in immunogenicity of O. niloticus GBS than does genotype, offering further information for design of a vaccine against O. niloticus GBS. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis is a causative agent of tuberculosis ( TB). The ability of M. tuberculosis to be quiescent in the cell has caused the emergence of latent infection. A comprehensive proteomic analysis of M. tuberculosis H37 Rv over three growth phases, namely mid-log (14-day culture), early stationary (28-day culture), and late stationary (50-day culture), was performed in order to study the change in proteome from the mid-log phase to late-stationary phase. Combination methods of two-dimensional electrophoresis (2- DE) and tandem mass spectrometry were used to generate proteome maps of M. tuberculosis at different growth phases. Ten proteins were detected differentially expressed in the late-stationary phase compared with the other two phases. These proteins were SucD, TrpD, and Rv2161c, which belong to metabolic pathway proteins; FadE5, AccD5, DesA1, and Rv1139c are proteins involved in cell wall or lipid biosynthesis, whereas TB21.7 and Rv3224 are conserved hypothetical proteins with unknown function. A surface antigen protein, DesA1, was not detectable in the late-stationary phase, although present in both log and early-stationary phases. The changes in the expression levels of these proteins were in line with the growth environment changes of the bacteria from mid-log phase to late-stationary phase. The information gathered may be valuable in the intervention against latent TB infection. [ABSTRACT FROM AUTHOR]

ABSTRACT In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h-1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water-insoluble proteins by two-dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween-80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60- kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55- kDa protein (p55; actin) and a 49- kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50- kDa protein (p50d; α-tubulin), a 25- kDa protein (p25; α-tubulin) and a 52- kDa protein (p52c; β-tubulin) was enhanced. [ABSTRACT FROM AUTHOR]

Proteomic analysis of plants relies on high yields of pure protein. In plants, protein extraction and purification present a great challenge due to accumulation of a large amount of interfering substances, including polysaccharides, polyphenols, and secondary metabolites. Therefore, it is necessary to modify the extraction protocols. A study was conducted to compare four protein extraction and precipitation methods for proteomic analysis. The results showed significant differences in protein content among the four methods. The chloroform-trichloroacetic acid-acetone method using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( HEPES) buffer provided the best results in terms of protein content, pellets, spot resolution, and intensity of unique spots detected. An overall of 83 qualitative or quantitative significant differential spots were found among the four methods. Based on the 2- DE gel map, the method is expected to benefit the development of high-level proteomic and biochemical studies of A ndrographis paniculata, which may also be applied to other recalcitrant medicinal plant tissues. [ABSTRACT FROM AUTHOR]

Protein extraction is a crucial step for proteomics studies. To establish an effective protein extraction protocol suitable for two-dimensional electrophoresis (2 DE) analysis in Jerusalem artichoke ( Helianthus tuberosus L.), three different protein extraction methods-trichloroacetic acid/acetone, Mg/ NP-40, and phenol/ammonium acetate-were evaluated using Jerusalem artichoke leaves as source materials. Of the three methods, trichloroacetic acid/acetone yielded the best protein separation pattern and highest number of protein spots in 2 DE analysis. Proteins highly abundant in leaves, such as Rubisco, are typically problematic during leaf 2 DE analysis, however, and this disadvantage was evident using trichloroacetic acid/acetone. To reduce the influence of abundant proteins on the detection of low-abundance proteins, we optimized the trichloroacetic acid/acetone method by incorporating a PEG fractionation approach. After optimization, 363 additional (36.2%) protein spots were detected on the 2 DE gel. Our results suggest that trichloroacetic acid/acetone method is a better protein extraction technique than Mg/ NP-40 and phenol/ammonium acetate in Jerusalem artichoke leaf 2 DE analysis, and that trichloroacetic acid/acetone method combined with PEG fractionation procedure is the most effective approach for leaf 2 DE analysis of Jerusalem artichoke. [ABSTRACT FROM AUTHOR]

ABSTRACT To investigate the late and persistent effects of cadmium (Cd) at low doses on the liver and its potential mechanisms, male Wistar rats were given i.p. injection of Cd as CdCl2 at 20 nmol kg−1 body weight every other day for 4 weeks. At weeks 20, 44 and 52, the livers from Cd-treated and age-matched control rats were examined pathologically and biochemically. Chronic exposure of rats to Cd at low doses induced mild pathological changes and persistent oxidative damage as well as cell proliferation. Hepatic proteins were analyzed with two-dimensional electrophoresis (2-DE) and mass spectrometry. More than 1000 protein spots were detected by 2-DE. Ten proteins were distinguishable between Cd-treated and age-matched control groups at week 52 week after Cd treatment. Two of them were significantly down-regulated: prohibitin (PHB) and d-dopachrome tautomerase (DDT). By western blotting the down-regulated expression of PHB and DDT in the livers of Cd-treated rats was confirmed in both early (week 20) and late (week 52) time points. To further examine the down-regulation of antioxidant status in the Cd-treated livers, other common antioxidants, including superoxide dismutase and glutathione and one metal detoxification specific protein metallothionein, were also detected and found to be decreased, particularly at the late stage. These results suggest that mild histopathological changes, persistent oxidative damage and cell proliferation remained at the late stages (weeks 44-52) after rats were exposed to low-dose Cd. These persistent changes may be associated with the persistent down-regulation of cellular antioxidant systems. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: Intrauterine growth restriction (IUGR) has been associated with increased perinatal morbidity, higher incidence of neurodevelopmental defects and increased risk for adult metabolic syndrome manifestations. Altered protein expression profiles associated with IUGR may be informative on the pathological mechanisms of this condition and might reveal potential markers for postnatal complications. We hypothesized that nutrient manipulation of the pregnant rat might influence the expression of important neurodevelopmental proteins in the resultant IUGR offspring. Therefore, we aimed to determine in newborn rat brain tissue the expression of collapsin response mediator proteins (CRMPs)-1, -2 and -5, commonly referred to as dihydropyrimidinase-related proteins (DPYLs) – playing a role in axon guidance, invasive growth and cell migration – and compare it to the corresponding expression in control rats. Two-dimensional electrophoresis and mass spectrometry, as well as Western blot analysis were employed in brain tissue from 24 IUGR newborn rats and 24 controls. With both methods, CRMP-1, CRMP-2 and CRMP-5 were decreased in the brains of the IUGR group as compared to the control group at the time of delivery. In conclusion, IUGR rat offspring are born with a decreased expression of CRMPs, suggesting that these proteins may be implicated in fetal stress-induced programming. [Copyright &y& Elsevier]

Wheat protein composition and organization play interrelated roles in determining physical properties for technological purposes. In prior research, a number of isogenic wheat lines of Bobwhite that have high levels of expression of the native Dx5 and/or Dy10 high‐molecular‐weight glutenin subunits (HMW‐GS) were examined vis‐à‐vis physical properties related to separation. In particular, these altered lines were characterized by poor mixing properties, the formation of mixtures in water that could not be separated by conventional mechanical methods, reduced water absorption, unique milling properties, and severely limited development of microscopic fibrils. These attributes suggested inherent organizational differences at submicroscopic and molecular levels among the various lines. Therefore, proteins were fractionated from whole meals using 70% ethanol to elucidate solubility characteristics and compositions and to infer structural properties. Capillary zone electrophoresis and one‐ and two‐dimensional SDS‐PAGE (2DE) revealed striking differences in the protein composition and solubility among these new lines and the Bobwhite from which they were derived. Generally, Bobwhite yielded soluble protein that included not only what would be considered as classical gliadins but also some of each of the HMW‐GS as monomers or polymers with low degrees of polymerization, whereas the genetically altered lines produced far less total soluble protein and very limited amounts of HMW‐GS. In the extreme, high levels of expression of Dx5 subunit not only led to reduced solubility of the HMW‐GS but also limited the solubility of the many other proteins that are normally soluble. In addition, a matrix association factor similar to the classical separation factor of analytical chemistry and chemical engineering was introduced and applied to 2DE data for insoluble and soluble protein to summarize and index relative involvement of specifically enhanced proteins in the insoluble gluten matrix after equilibration with ethanol. The highest relative association was determined for the HMW‐GS lines enriched in Dx5 or Dy10 protein and the lowest for Bobwhite. Greater association was indicated for Dx5 than for Dy10 protein in these lines. The value of the association factor was likely influenced by differences in glutamine‐to‐cysteine ratios and differences in altered glutenin chain configurations stemming from high levels of expression of a single or limited number of HMW‐GS. [ABSTRACT FROM AUTHOR]

Potato can be severely affected by various pathogens, including Pectobacterium atrosepticum, the cause of bacterial soft rot on tubers and of blackleg on stems. To date, no complete resistance to P. atrosepticum is available, so that only cultivars exhibiting partial resistance can be found. The mechanistic basis of this type of resistance is still poorly understood. A proteomic approach was thus developed to identify pathways specifically activated during the interaction between potato tubers and P. atrosepticum. Protein profiles on silver-stained gels in the 5-8 p H range were obtained from healthy and infected tubers from two cultivars differing for resistance level and analyzed by 2- DE and nano- LC- MS/ MS. Thirteen proteins were differentially up-regulated in the partially resistant cv. Kerpondy; by contrast, no significant differences in protein profiles of inoculated and control tubers were observed in the susceptible cv. Bintje. Mass spectrometry and database searching showed that these proteins are involved in energetic metabolism (glyceraldehyde-3-phosphate dehydrogenase, 2-phosphoglycerate dehydratase or enolase, fructose biphosphate aldolase and ATPase α subunit), cytoskeleton structure (actin), protein catabolism (cysteine protease inhibitor) and patatins or patatin precursors. Their involvement in defence responses of cv. Kerpondy to P. atrosepticum is discussed. Proteomic appears as an efficient approach to have insight into the mechanisms and pathways leading to potato resistance against P. atrosepticum. [ABSTRACT FROM AUTHOR]

SUMMARY The effects of the cruciferous phytoalexin brassinin on the protein expression patterns of the phytopathogenic fungus Alternaria brassicicola were investigated. Cell-free protein extracts of mycelia of A. brassicicola induced with brassinin at 0.50 and 0.10 m m were fractionated, and the proteins in soluble fractions were separated by two-dimensional electrophoresis. Spots corresponding to differentially expressed proteins were digested and analysed by liquid chromatography-electrospray ionization-mass spectrometry. The number of differentially expressed proteins was significantly higher in mycelia treated with brassinin at 0.50 m m (96 protein spots) than in mycelia treated with brassinin at 0.10 m m (18 protein spots). The majority of differentially expressed proteins included proteins involved in metabolism, processing, synthesis and several heat shock proteins (HSPs). Brassinin concentrations below 0.30 m m induced HSP90, a protein involved in the regulation of morphogenetic signalling in fungi, suggesting that 0.30 m m is a minimal concentration of brassinin necessary for the protection of brassicas against A. brassicicola. These results reveal that HSP90 is a potential target for inhibition in stressed A. brassicicola and confirm that brassinin has strong detrimental effects on A. brassicicola, suggesting that its detoxification by the fungus suppresses an important defence layer of the plant. [ABSTRACT FROM AUTHOR]