Protein Spots Research Articles

Comparison of Five TRIzol-Based Protein Preparation Methods for 2-DE Production From Challenging Marine Dinoflagellate Samples: A Case Study on Two Benthic Prorocentrum Species

Two-dimensional gel electrophoresis (2-DE) is a major element of conventional gel-based proteomics, which resolves complex protein mixtures. Protein extraction with the removal of interfering substances from the sample remains the key to producing high-quality 2-DE profiles. Marine dinoflagellates contain large endogenous amounts of salts, nucleic acids, polysaccharides, phenolic compounds, pigments, and other interfering compounds. These substances are detrimental to the quality of gel images. Protein preparation using TRIzol reagent is a promising method for producing high-quality 2-DE profiles for dinoflagellate samples. In addition to its remarkable performance, the TRIzol method’s several advantages have made it a popular and widely used method in the field of 2-DE sample preparation. Nonetheless, the quality of 2-DE of samples from certain dinoflagellate species is not as high as previously reported when the same TRIzol protocol is applied. Therefore, modifications to the original TRIzol method are required to remove interfering substances from those challenging dinoflagellate samples. In this study, the original TRIzol method and four modified methods, namely the aliquot TRIzol method, re-TRIzol method, TRIzol method with a commercial clean-up kit, and TRIzol method with trichloroacetic acid/acetone precipitation, were compared. Performance of these five methods in terms of protein yield, background signal, and resolution and number of protein spots was investigated on samples from two benthic Prorocentrum species: P. lima and P. hoffmannianum. Our results demonstrated that high-quality 2-DE could be achieved from P. lima samples prepared using both the original TRIzol method and the TRIzol method with a commercial clean-up kit. However, the original TRIzol method failed to produce high-quality 2-DE profiles for P. hoffmannianum samples. Among the four modified TRIzol methods, only the TRIzol method with a commercial clean-up kit could yield substantially improved high-quality 2-DE profiles for P. hoffmannianum samples. This combination of the conventional TRIzol method with a commercial clean-up kit potentially represents a promising protein extraction methodology for obtaining high-quality 2-DE profiles for difficult dinoflagellate samples.

A comparative study of the accuracy of the spot Protein-Creatinine Ratio (PCR) vs 24 hours urine collection for detection of proteinuria in pre- eclamptic patients and its effect maternal and fetal outcomes

Proteinuria is a major component of preeclampsia. Urine protein measurement after 24-hour urine collection is the traditional standard method for the detection of proteinuria. It is time-consuming. As an alternative, spot sampling for a urine protein to creatinine ratio (PCR) ratio has been investigated. To determine the accuracy of the spot protein- creatinine ratio in comparison with 24 hours urine collection for detection of proteinuria in pre- eclamptic patients. A hospital based prospective study was conducted with 120 patients to assess the quantification of proteinuria in preeclamptic patient and comparing the two methods i.e. 24-hr urine protein levels and random protein creatinine ratio conducted at tertiary care centre. There was a good positive statistical correlation between 24-hour total urinary protein level and spot protein: creatinine ratio with a correlation coefficient (r) of 0.769.Conclusion: Random and spot urine protein creatinine ratio can predict with high accuracy the amount of 24- hour urine protein excretion. The urine PCR is reliable, relatively faster and accurate for proteinuria and can correlate well with 24-hour urinary protein excretion which is gold standard.

Proteomic analysis of the inhibitory effect of the butanolic fraction of Jacquinia macrocarpa on Fusarium verticillioides.

Jacquinia macrocarpa, a plant native to northwestern Mexico, has an inhibitory effect against phytopathogenic fungi. Previous studies have shown that the butanolic extract of J. macrocarpa causes retardation and atrophy in mycelial growth of Fusarium verticillioides. However, the action mechanism of this extract is unknown. We used a proteomics approach to understand the inhibitory effect of J. macrocarpa butanolic extract, based on differential protein accumulation in F. verticillioides. Proteins were extracted from F. verticillioides cultured in Czapek broth with and without 202.12 μg/mL (IC50) of butanolic extract of J. macrocarpa. Thirty-eight protein spots showing statistically significant changes (ANOVA, p < 0.01) and at least a 2-fold change in abundance between experimental conditions were analyzed by mass spectrometry. Identified proteins were grouped into different biological processes according to Gene Ontology, among them were amino acid metabolism, protein folding and stabilization, protein degradation, protein transport, carbohydrate metabolism, oxidative stress response, and miscellaneous. This work is the first report of changes in the proteomic profile of F. verticillioides exposed to the J. macrocarpa extract. This information provides new insights into the inhibitory mechanism of the extract and represents a starting point for dissection of the fungal response against the J. macrocarpa extract components.

Improved Alignment and Quantification of Protein Signals in Two-Dimensional Western Blotting.

Western blotting is widely used for protein identification and quantification in research applications, but different protein species, resulting from alternative splicing and post-translational modifications, can often only be detected individually by two-dimensional gel electrophoresis and immunodetection by Western blotting (2D-WB). The additional separation by isoelectric focusing enables the detection of different protein species with the same specific antibody. Reliable assignment of signals from antibody-based detection to the total protein spot pattern of the original gel image is a challenge in 2D-WB, often resulting in ambiguous results. We therefore propose a reliable strategy for assignment of antibody signals from 2D-WB to the total protein spot pattern, using an imaging workflow in combination with a straightforward and easily reproducible image alignment strategy. The strategy employs vector-based alignment of protein spots and image contours in a stepwise manner. Our workflow is compatible with various protein visualization techniques, including prelabeling of proteins and poststaining of gels and membranes, as well as with chemiluminescent and fluorescent detection of bound antibody. Here, we provide a detailed description of potential applications and benefits of our workflow. We use experimental test settings with gold-standard stressors in combination with multiple staining and detection methods, as well as spike-in recombinant proteins. Our results demonstrate reliable attribution of signals to very similar heat shock proteins, phosphorylation patterns, and global analysis of proteins modified with O-linked N-acetylglucosamine (O-GlcNAc).

Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences After Differential Ultracentrifugation and ExoQuickTM Isolation.

Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuick™ in parallel. EV associated protein profiles were compared by multiplex-fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and subsequent identification by mass spectrometry. Validation of gelsolin (GSN) was performed using fluorescence-quantitative western blot. 2D-DIGE resolved 936 protein spots in all serum-enriched EVs isolated by ultracentrifugation or ExoQuick™. Hereof, 93 spots were differently expressed between isolation approaches. Higher levels of GSN in EVs obtained with ExoQuick™ compared to ultracentrifugation were confirmed by western blot (p = 0.0006). Although patient groups were distinguishable after both EV isolation approaches, sample preparation strongly influences EVs’ protein profile and thus impacts on inter-study reproducibility, biomarker identification and validation. The results stress the need for strict SOPs in EV research before clinical implementation can be reached.

Dynamic proteomic analysis of Phanerochaete chrysosporium under copper stress.

The model white rot fungus Phanerochaete chrysosporium is frequently preferred for heavy metal accumulation studies due to its high resistance to heavy metals, including copper (Cu). Here, the response of P. chrysosporium under Cu stress at different time points was investigated for the first time by a detailed proteomic analysis using 2DE MALDI-TOF/MS and nanoLC-MS/MS techniques. A total of 123 Cu-responsive protein spots were determined using 2DE approach, and 104 of them were corresponded to 73 distinct open reading frames (ORFs). Of identified ones, 88 spots were over-, and 16 spots were underrepresented. The majority of these proteins showed to the strongest response at 8th h of Cu exposure. Using nanoLC-MS/MS analysis, a total of 167 differentially produced proteins were identified from Cu-exposed cultures after enrichment of the membrane proteins followed by SILAC. Seventy four, 66, and 69 overrepresented, and 56, 71, and 64 underrepresented proteins were identified at 2h, 4h, and 8h of Cu exposure, respectively. The bioinformatic analysis of these proteins revealed that intracellular trafficking proteins such as Ran GTPase and a p24 family protein, and certain proteins involved in posttranslational modification, protein turnover and folding were Cu-responsive. Three important transcription factors (TFs), NAC, BTF3, and homeobox TFs, 40S and 60S ribosomal proteins, chaperones such as Hsp26/Hsp42 and mortalin, as well as 20S proteasome, 14-3-3 proteins and Hsp90 involve in Cu-stress response of P. chrysosporium. Moreover, certain elements of translation machinery, the proteins related with aspartate, methionine, and pyruvate metabolisms, transketolase, and trehalase related with carbohydrate metabolism, citrate synthase, fumarase, V-ATPase, and F0F1-type ATPase playing role in energy production and conversion, transport proteins such as multidrug resistance and p24 family proteins as well as actin-related proteins involved in cytoskeleton remodeling were determined to be Cu-responsive. The present proteome analysis revealed that P. chrysosporium mainly regulates translational and posttranslational processes, certain transport processes, many metabolic pathways and cytoskeleton to overcome the Cu-induced oxidative stress.

Comparative assessment of proteins between cell lines and tissues representing carcinomas of the breast using 2-dimensional gel electrophoresis

Incidence of breast cancer in the age group of 30 to 50 is increasing at an alarming rate globally. Moreover, existing treatments are either marginally effective or resistance developed, hence development of safe and potent pharmacological agents is immediately required. However, establishment of a disease progression marker for early detection, development of safe and effective treatment agents requires identification of key proteins that are exclusively expressed in advanced malignant breast tumors. 2-Dimensional gel electrophoresis (2-DGE) is one of the early methods used for the identification of deregulated proteins in cancers. Therefore, the proteins of benign andmalignant tumors as well as breast cancer cell lines MCF-7, SKBR-3 and MDAMB- 468, were subjected to 2-DGE and relative expression calculated. Analysis of the data showed a significant increase in the intensity of 7 protein spots compared to malignant tissues and benign ones. Further, comparison of cell line proteins with tissue lysates revealed that SKBR-3 is much closer to malignant tumors, hence, may be considered for screening drug targets as well as for evaluating the efficacy of pharmacological agents. In conclusion, our 2-DGE identified key differences and similarities in the expression of proteins between breast cancer cell lines, benign and malignant tissues.

Comparative proteomics study of proteins involved in induction of higher rates of cell death in mitoxantrone-resistant breast cancer cells MCF-7/MX exposed to TNF-α.

Objective(s):Resistance to medications is one of the main complications in chemotherapy of cancer. It has been shown that some multidrug resistant cancer cells indicate more sensitivity against cytotoxic effects of TNF-α compared to their parental cells. Our previous findings indicated vulnerability of the mitoxantrone-resistant breast cancer cells MCF-7/MX to cell death induced by TNF-α compared to the parent cells MCF-7. In this study, we performed a comparative proteomics analysis for identification of proteins involved in induction of higher susceptibility of MCF-7/MX cells to cytotoxic effect of TNF-α.Materials and Methods:Intensity of protein spots in 2D gel electrophoresis profiles of MCF-7 and MCF-7/MX cells were compared with Image Master Platinum 6.0 software. Selected differential protein-spots were identified with MALDI-TOF/TOF mass spectrometry and database searching. Pathway analyses of identified proteins were performed using PANTHER, KEGG PATHWAY, Gene MANIA and STRING databases. Western blot was performed for confirmation of the proteomics results.Results:Our results indicated that 48 hr exposure to TNF-α induced 87% death in MCF-7/MX cells compared to 19% death in MCF-7 cells. Forty landmarks per 2D gel electrophoresis were matched by Image Master Software. Six proteins were identified with mass spectrometry. Western blot showed that 14-3-3γ and p53 proteins were expressed higher in MCF-7/MX cells treated with TNF-α compared to MCF-7 cells treated with TNF-α.Conclusion:Our results showed that 14-3-3 γ, prohibitin, peroxiredoxin 2 and P53 proteins which were expressed differentially in MCF-7/MX cells treated with TNF-α may involve in the induction of higher rates of cell death in these cells compared to TNF-α-treated MCF-7 cells.

Transformation of hybrid black poplar with selective and reporter genes affects leaf proteome, yet without indication of a considerable environmental hazard

Poplar is an economically important forest tree used for multiple applications, including bioenergy production. Although genetic engineering allows a quick introduction of desirable traits, simultaneously, it may deliver unintended side effects. Herein, we compared the protein composition in leaves of microclonally propagated fast-growing hybrid clone of Populus deltoides × Populus nigra (Populus × euroamericana), genetically modified with a reporter (GUS) and selective (NPTII) traits, and its non-transgenic parental genotype. By two-dimensional gel electrophoresis, we revealed that 5.5% of 330 detected protein spots were differentially accumulated. The vast majority of them showed lower abundance in the transgenic line. These proteins were mainly involved in primary metabolism, energy production, and protein synthesis/homeostasis; notably, none of them was directly linked to neomycin phosphotransferase or β-glucuronidase activities. Some of the differentially accumulated proteins could be associated with stress response caused by Agrobacterium-mediated transformation. Furthermore, the lower abundance of two cytosolic glutamine synthase isoforms and accumulation of bark storage protein A in the transgenic line, plausibly indicated impaired nitrogen metabolism and storage. The insertion of a model genetic construct did not alter the neighboring genetic loci, and neither toxins nor allergens were differentially abundant. In essence, we detected no protein indicating considerable health or environmental hazard in the transgenic hybrid black poplar.

Defense Response in Chickpea Pod Wall due to Simulated Herbivory Unfolds Differential Proteome Profile.

The pod wall of legumes is known to protect the developing seeds from pests and pathogens. However, the mechanism of conferring defense against insects has not yet been deciphered. Here, we have utilized 2-dimensional gel electrophoresis (2D-GE) coupled with mass spectrometry (MS/MS) to identify over expressed proteins in the pod wall of two different cultivars (commercial cultivar: JG 11 and tolerant cultivar: ICC 506-EB) of chickpea after 12h of application of Helicoverpa armigera oral secretions (simulated herbivory). The assays were performed with a view that larvae are a voracious feeder and cause substantial damage to the pod within 12h. A total of 600 reproducible protein spots were detected on gels, and the comparative analysis helped identify 35 (12 up-regulated, 23 down-regulated) and 20 (10 up-regulated, 10 down-regulated) differentially expressed proteins in JG 11 and ICC 506-EB, respectively. Functional classification of protein spots of each cultivar after MS/MS indicated that the differentially expressed proteins were associated with various metabolic activities. Also, stress-related proteins such as mannitol dehydrogenase (MADH), disease resistance-like protein-CSA1, serine/threonine kinase (D6PKL2), endoglucanase-19 etc. were up-regulated due to simulated herbivory. The proteins identified with a possible role in defense were further analyzed using the STRING database to advance our knowledge on their interacting partners. It decoded the involvement of several reactive oxygen species (ROS) scavengers and other proteins involved in cell wall reinforcement. The biochemical analysis also confirmed the active role of ROS scavengers during simulated herbivory. Thus, our study provides valuable new insights on chickpea-H.armigera interactions at the protein level.

Emerging Allergens in Goji Berry Superfruit: The Identification of New IgE Binding Proteins towards Allergic Patients' Sera.

The goji berry (Lycium barbarum L.) (GB) is gaining increasing attention with high consumption worldwide due to its exceptional nutritional value and medicinal benefits displayed in humans. Beyond their beneficial properties, GBs contain renowned allergenic proteins, and therefore deserve inclusion among the allergenic foods capable of inducing allergic reactions in sensitive consumers. GB allergy has been frequently linked to the panallergen lipid transfer protein (LTP), especially across the population of the Mediterranean area. Methods: In this study, we investigated the protein profile of GBs focusing on the most reactive proteins against immunoglobulins E (IgE) of allergic patients’ sera, as ascertained by immunoblot experiments. The protein spots displaying a clear reaction were excised, in-gel digested, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by data searching against a restricted database for a reliable protein identification. Results: According to our data, three main spots were identified in GB extract as IgE binding proteins after immunoblot analysis. Some major proteins were identified and the three proteins that provided the highest reactivity were putatively attributed to vicilin and legumin proteins followed by a protein matching with 11S globulin belonging to the cupin superfamily. Finally, the whole GB protein extract was also submitted to bottom-up proteomics followed by a software-based database (DB) screening and a more exhaustive list of GB proteins was compiled.

Appropriate Ammonium/Nitrate Mitigates Low Light Stress in Brassica pekinensis by Regulating the Nitrogen Metabolism and Expression Levels of Key Proteins

Low light intensity is always the important factor that influences the growth and development of Brassica pekinesis during winter seasons. We have found out that appropriate ammonium/nitrate ratio mitigated low light intensity in the previous study. In the present study, the nitrogen metabolism and proteomic responses of Brassica pekinensis to light intensities and ammonium/nitrate were further investigated to find out the mitigation mechanism. The activities of nitrate reductase (NR) and glutamine synthetase (GS) and the relative expression of GS1 as well as the content of nitric oxide (NO) were significantly higher in the NH4+:NO3− (15:85) treatment than in the NH4+:NO3− (0:100) treatment under normal light intensity. However, under low light intensity condition, the activities of NR and GS and relative expression of NR as well as the NO level in seedlings fertilized with NH4+:NO3− (10:90) were significantly higher compared with those fertilized with NH4+:NO3− (0:100). In addition, we found thirty-six protein spots, which exhibited significant changes in abundance using similarity searches across the uniprot database of Brassica. The proteins identified were classified into ten functional groups including photosynthesis, carbon and energy metabolism, stress/defense, protein folding, and modification and degradation response. We concluded that appropriate ammonium nitrate ratio improves the tolerance of mini Chinese cabbage seedlings to low light intensity by regulating the nitrogen metabolism and expression levels of some key proteins.

DPYSL2 is a novel regulator for neural stem cell differentiation in rats: revealed by Panax notoginseng saponin administration

BackgroundThe limited neuronal differentiation of the endogenous or grafted neural stem cells (NSCs) after brain injury hampers the clinic usage of NSCs. Panax notoginseng saponins (PNS) were extensively used for their clinical value, such as in controlling blood pressure, blood glucose, and inhibiting neuronal apoptosis and enhancing neuronal protection, but whether or not it exerts an effect in promoting neuronal differentiation of the endogenous NSCs is completely unclear and the potential underlying mechanism requires further exploration.MethodsFirstly, we determined whether PNS could successfully induce NSCs to differentiate to neurons under the serum condition. Mass spectrometry and quantitative polymerase chain reaction (Q-PCR) were then performed to screen the differentially expressed proteins (genes) between the PNS + serum and serum control group, upon which dihydropyrimidinase-like 2 (DPYSL2), a possible candidate, was then selected for the subsequent research. To further investigate the actual role of DPYSL2 in the NSC differentiation, DPYSL2-expressing lentivirus was employed to obtain DPYSL2 overexpression in NSCs. DPYSL2-knockout rats were constructed to study its effects on hippocampal neural stem cells. Immunofluorescent staining was performed to identify the differentiation direction of NSCs after 7 days from DPYSL2 transfection, as well as those from DPYSL2-knockout rats.ResultsSeven differentially expressed protein spots were detected by PD Quest, and DPYSL2 was found as one of the key factors of NSC differentiation in a PNS-treated condition. The results of immunostaining further showed that mainly Tuj1 and GFAP-positive cells increased in the DPYSL2-overexpressed group, while both were depressed in the hippocampal NSCs in the DPYSL2-knockout rat.ConclusionsThe present study revealed that the differentiation direction of NSCs could be enhanced through PNS administration, and the DPYSL2 is a key regulator in promoting NSC differentiation. These results not only emphasized the effect of PNS but also indicated DPYSL2 could be a novel target to enhance the NSC differentiation in future clinical trials.

Proteomic Characterisation of Lupin (Lupinus angustifolius) Milk as Influenced by Extraction Techniques, Seed Coat and Cultivars

Lupin seeds are rich in proteins and other essential ingredients that can help to improve human health. The protein contents in both whole and split seeds of two lupin cultivars (Mandleup and PBA Jurien) were used to produce the lupin milk using the cheesecloth and centrifuge method. Proteins were extracted from the lupin milk using thiourea/urea solubilization. The proteins were separated by a two-dimensional polyacrylamide gel electrophoresis and then identified with mass spectrometry. A total of 230 protein spots were identified, 60 of which showed differential abundances. The cheesecloth separation showed protein extractability much better than that of the centrifuge method for both the cultivars. The results from this study could offer guidance for future comparative analysis and identification of lupin milk protein and provide effective separation technique to determine specific proteins in the cheese-making process.

Immuno-proteomics analysis between OMV of vaccine and dominant wild type strains of Bordetella pertussis in Iran

Despite widespread vaccination programs against pertussis, there has been a worldwide resurgence of the disease in recent years. We aimed to investigate protein composition of outer membrane vesicles (OMV) of Bordetella pertussis (Bp) and to evaluate the immunogenicity of OMV antigens both in the vaccine and the dominant wild type strains in Iran. The OMV were purified from both vaccine and wild type strains. The immunoreactivity of the OMVs was investigated by exposing sera taken from the patients and the vaccinated infants. The protein profiles of OMVs were compared using two-dimensional electrophoresis. The LC-MS/MS was used to analyse and identify differentially expressed protein spots. The two type strains showed differences in their 2D gel protein profile. Further analysis of selected proteins from the dominant Iranian strains using LC-MS/MS demonstrated that the identified proteins fell into different functional categories including (i) metabolism, (ii) membrane transport and secretion system, (iii) biosynthesis and degradation, (iv) adaption, adhesion, pathogenicity, conserved hypothetical and protection responses. Moreover, a number of immunogenic proteins were identified including Bp 2434 (serine protease) and Bp 1616 (putative DNA binding protein) from the vaccine and the wild type strains, respectively which could be considered as potential antigens for an OMV vaccine. OMV Bp could be considered as an alternative vaccine against pertussis, containing the bacterium's protein antigens that can confer equal efficacy compared to a whole bacterial cell vaccine with advantages such as less side effects and lower costs than acellular pertussis vaccines.

Differential methods of cell lysis for protein profiling of Alexandrium sp. using 2D gel electrophoresis

Introduction: Protein profiling of harmful algae is an ongoing study where the latest analysis was conducted on A. minutum. It is a fundamental study where the protein expression of targeted species can be used to understand the biochemical pathway of selected proteins. The Malaysia Alexandrium spp. has the potential to cause massive blooming that brings harm to the aquatic ecosystem. Methods: In this experiment three methods of cell lysis were tested against A. leei isolated from Malaysian waters. Results: The Axenic culture of the sample was established in enriched seawater media (ESDK) with 12 hours of light and 12 hours of dark conditions. The sample was extracted during exponential phase (day 18) where the same amount of cells was collected via centrifugation. The same buffer was used for each technique of cell lysis in order to get the best protein profiles in terms of a band or spot intensity and number. The cells of A. leei were lysed using sonication in the ice-cold water bath, sonication with probe and freeze-thawing followed by sonication in iced bath is the best method of protein extraction. Conclusions: Nevertheless, it can be concluded that the qualitative analysis using SDS-PAGE and 2D gel electrophoresis could be best method and the freeze thawing mode of cell lysis produced an excellent result among others as the protein spots produced were precise and less streaking were observed.

Protein profiling of chicken breast muscles from different slaughter houses in relation to meat quality

Introduction: Chicken meat is a source of protein in the human diet. Protein content and values define the quality of chicken meat. This research aimed to analyze variations of protein profiles in chicken breast muscles from different slaughtering houses by using proteomic strategies. Methods: Total proteins of chicken breast muscles from three different slaughtering houses (Sample A, Sample B and Sample C) were extracted and quantified by using Bradford assay. Then, the proteins were separated by SDS-PAGE to monitor the quality of extracted proteins. Protein profiles in different samples were compared by 2D-GE analysis. Results: The most highly expressed protein band was located between the molecular size of 37-50 kD in all samples and it was expected to be betaactin. While resolved in 2D-PAGE, differences in protein expression were observed between samples. There were three spots expressed with highest intensity in Sample B compared to others. The protein spot detected at pH 5.28 and the size range between 50- 75 kD was predicted to be NADPH-cytochrome P450 reductase (CPR), at pH 6.45 and molecular weight between 37-50 kD was expected to be creatine kinase M-type (M-CK) while at pH 6.78 and molecular size nearly 25 kD was expected to be αB-crystallin. Conclusions: It can be mentioned that these proteins could play a vital role in mechanisms that contribute to the poor quality of chicken meat.

Changes in Salivary Proteome in Response to Bread Odour.

It is widely recognized that smelling food results in a mouth-watering feeling and influences appetite. However, besides changes in volume, little is known about the effects that food odours have on the composition of saliva. The aim of the present study was to access the effects that smelling bread has on saliva proteome and to compare such effects with those of chewing and ingesting it. Besides a significant increase in saliva flow rate, together with a decrease in total protein concentration, bread odour induced changes in the proportion of different salivary proteins. The expression levels of two spots of cystatins and two spots of amylase increased due to olfactory stimulation, similar to what happened with bread mastication, suggesting that odour can allow anticipation of the type of food eaten and consequently the physiological oral changes necessary to that ingestion. An interesting finding was that bread odour increased the expression levels of several protein spots of immunoglobulin chains, which were decreased by both bread or rice mastication. This may be of clinical relevance since food olfactory stimulation of salivary immunoglobulins can be used to potentiate the oral immune function of saliva. Moreover, the effects of bread odour in the levels of salivary proteins, previously observed to be involved in oral food processing led to the hypothesis of an influence of this odour in the sensory perception of foods further ingested. Further studies are needed to elucidate this point, as well as whether the changes observed for bread odour are specific, or if different food odours lead to similar salivary proteome responses.

Leaf proteome pattern of two bread wheat varieties under water deficit stress conditions

This investigation was carried out to study the effect of water deficit stress on leaf proteome pattern and agronomic and physiological characteristics of two spring bread wheat varieties (Arg and Moghan3) with contrasting responses to drought stress. For this purpose, an experiment was conducted as a 2 × 2 factorial arranged as the randomized complete block design with five replications in a greenhouse of University of Tabriz, Iran. The factors consisted of two Arg and Moghan3 varieties and irrigation conditions (normal condition and drought stress, where water was withheld at the pollination stage). Based on the analysis of variance, significant differences were observed between two wheat varieties with respect to all studied characters except leaf chlorophyll content. Drought stress increased leaf temperature and reduced other characters. Leaf proteome analysis was performed by two-dimensional electrophoresis for both wheat varieties at two irrigation conditions. After quantification of spots with PDQuest software, 274 and 202 repeatable protein spots were identified in Arg and Moghan3 varieties, respectively. Among them, based on the induction factor and t-test, 73 and 64 protein spots showed significant changes in abundance in Arg and Moghan3 varieties, respectively. Out of 73 spots identified in Arg, 46 showed an increased expression and 27 showed a decreased expression. In Moghan3, out of 64 protein spots, 18 and 46 protein spots were up- and down-regulated, respectively. Moreover, 15 common protein spots in Arg and Moghan3 with significant changes in expression between normal and drought stress conditions were identified by applying MALDI-TOF/TOF mass spectrometry. Identified proteins were assigned to seven functional categories: photosynthesis, stress, defense and detoxification, glycolysis, transport, protein synthesis and transcription regulation. In general, proteins related to photosynthesis, stress, and defense and detoxification showed major changes which may be considered as important proteins contributing to drought tolerance of Arg in this study.

A proteomic approach towards understanding crypoprotective action of Me2SO on the CHO cell proteome

Chinese hamster ovary (CHO) cell lines are the most widely used in vitro cells for research and production of recombinant proteins such as rhGH, tPA, and erythropoietin. We aimed to investigate changes in protein profiles after cryopreservation using 2D-DIGE MALDI-TOF MS and network pathway analysis. The proteome changes that occur in CHO cells between freshly prepared cells and cryopreserved cells with and without Me2SO were compared to determine the key proteins and pathways altered during recovery from cryopreservation. A total of 54 proteins were identified and successfully matched to 37 peptide mass fingerprints (PMF). 14 protein spots showed an increase while 23 showed decrease abundance in the Me2SO free group compared to the control. The proteins with increased abundance included vimentin, heat shock protein 60 kDa, mitochondrial, heat shock 70 kDa protein 9, protein disulfide-isomerase A3, voltage-dependent anion-selective channel protein 2. Those with a decrease in abundance were myotubularin, glutathione peroxidase, enolase, phospho glyceromutase, chloride intracellular channel protein 1. The main canonical functional pathway affected involved the unfolded protein response, aldosterone Signaling in Epithelial Cells, 14-3-3-mediated signaling. 2D-DIGE MALDI TOF mass spectrometry and network pathway analysis revealed the differential proteome expression of FreeStyle CHO cells after cryopreservation with and without 5% Me2SOto involve pathways related to post-translational modification, protein folding and cell death and survival (score = 56, 22 focus molecules). This study revealed, for the first time to our knowledge the proteins and their regulated pathways involved in the cryoprotective action of 5% Me2SO. The use of 5% Me2SO as a cryoprotectant maintained the CHO cell proteome in the cryopreserved cells, similar to that of fresh CHO cells.