Mascot Software Research Articles

Purification and Biochemical Characterization of Novel Galectin from the Black Poplar Medicinal Mushroom Cyclocybe cylindracea (Agaricomycetes) Strain MEST42 from Algeria.

In the present study, a new galectin designated Cyclocybe cylindracea lectin (CCL) was extracted from the fruiting bodies of the wild black popular mushroom C. cylindracea grown in Algeria. The protein was isolated using sepharose 4B as affinity chromatography matrix, and galactose as elutant. The purified galectin was composed of two subunits of 17.873 kDa each, with a total molecular mass of 35.6 kDa. Its agglutinant activity was impeded by galactose and its derivatives, as well as melibiose. Lactose showed the highest affinity, with a minimal inhibitory concentration of 0.0781 mM. CCL was sensitive to extreme pH conditions, and its binding function decreased when incubated with 10 mM EDTA, and it could be restored by metallic cations such as Ca2+, Mg2+, and Zn2+. CCL agglutinated human red blood cells, without any discernible specificity. Circular dichroism spectra demonstrated that its secondary structure contained β-sheet as dominant fold. In addition, bioinformatics investigation on their peptide fingerprint obtained after MALDI-TOF/TOF ionization using mascot software confirmed that CCL was not like any previous purified lectin from mushroom: instead, it possessed an amino acid composition with high similarity to that of the putative urea carboxylase of Emericella nidulans (strain FGSC A4/ATCC 38163/CBS 112.46/NRRL 194/M139) with 44% of similarity score.

Preparation, purification, and identification of novel antioxidant peptides from red-bellied pacu (Piaractus brachypomus) fish meat protein hydrolysate.

The current study investigates the preparation, purification, and identification of novel antioxidant peptides from Piaractus brachypomus fish (RBPF) meat. Antioxidant peptides from RBPF meat protein hydrolysate (RPMPH) were fractionated by ultrafiltration (3kDa MWCO membrane). RPMPH-IF (MW < 3kDa) fraction displayed significantly higher antioxidant activities (P < 0.05) (DPPH, ABTS, FRAP, and Fe2+chelating activity). RPMPH-IF was purified by Sephadex G-25 gel filtration chromatography, and the RPMPH-1 fraction exhibited significantly higher antioxidant activities (P < 0.05). Subsequently, the RPMPH-1 fraction was purified by reversed-phase high-performance liquid chromatography. RPH-8 showed the highest antioxidant activities. The sequence of peptides of the RPH-8 fraction was later identified by LC-MS/MS and MASCOT software. RPH-8 fraction showed the two peptides with MW of 1105.52 Da and 748.25 Da, and the sequence of peptides was identified as His-Asn-Leu-Gly-Leu-Leu-His-Gly-Asp-Met and Asp-Ala-Pro-Ser-Met-Asn-Asp, respectively. Thus, RPMPH or purified antioxidant peptides produced by probiotic Bacillus strain could be a bio-functional ingredient in food and nutraceutical applications.

Urinary Protein Profiling for Potential Biomarkers of Chronic Kidney Disease: A Pilot Study.

Proteinuria is a risk factor for chronic kidney disease (CKD) progression and associated complications. However, there is insufficient information on individual protein components in urine and the severity of CKD. We aimed to investigate urinary proteomics and its association with proteinuria and kidney function in early-stage CKD and in healthy individuals. A 24 h urine sample of 42 individuals (21-CKD and 21-healthy individuals) was used for mass spectrometry-based proteomics analysis. An exponentially modified protein abundance index (emPAI) was calculated for each protein. Data were analyzed by Mascot software using the SwissProt database and bioinformatics tools. Overall, 298 unique proteins were identified in the cohort; of them, 250 proteins belong to the control group with median (IQR) emPAI 39.1 (19−53) and 142 proteins belong to the CKD group with median (IQR) emPAI 67.8 (49−117). The level of 24 h proteinuria positively correlated with emPAI (r = 0.390, p = 0.011). The emPAI of some urinary proteomics had close positive (ALBU, ZA2G, IGKC) and negative (OSTP, CD59, UROM, KNG1, RNAS1, CD44, AMBP) correlations (r < 0.419, p < 0.001) with 24 h proteinuria levels. Additionally, a few proteins (VTDB, AACT, A1AG2, VTNC, and CD44) significantly correlated with kidney function. In this proteomics study, several urinary proteins correlated with proteinuria and kidney function. Pathway analysis identified subpathways potentially related to early proteinuric CKD, allowing the design of prospective studies that explore their response to therapy and their relationship to long-term outcomes.

Global proteomic profiling of multiple organs of cats (Felis catus) and proteome-transcriptome correlation during acute Toxoplasma gondii infection

BackgroundToxoplasma gondii is a protozoan parasite which can infect almost all warm-blooded animals and humans. Understanding the differential expression of proteins and transcripts associated with T. gondii infection in its definitive host (cat) may improve our knowledge of how the parasite manipulates the molecular microenvironment of its definitive host. The aim of this study was to explore the global proteomic alterations in the major organs of cats during acute T. gondii infection.MethodsiTRAQ-based quantitative proteomic profiling was performed on six organs (brain, liver, lung, spleen, heart and small intestine) of cats on day 7 post-infection by cysts of T. gondii PRU strain (Genotype II). Mascot software was used to conduct the student’s t-test. Proteins with P values < 0.05 and fold change > 1.2 or < 0.83 were considered as differentially expressed proteins (DEPs).ResultsA total of 32,657 proteins were identified in the six organs, including 2556 DEPs; of which 1325 were up-regulated and 1231 were down-regulated. The brain, liver, lung, spleen, heart and small intestine exhibited 125 DEPs, 463 DEPs, 255 DEPs, 283 DEPs, 855 DEPs and 575 DEPs, respectively. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of all proteins and DEPs in all organs showed that many proteins were enriched in binding, cell part, cell growth and death, signal transduction, translation, sorting and degradation, extracellular matrix remodeling, tryptophan catabolism, and immune system. Correlations between differentially expressed proteins and transcripts were detected in the liver (n = 19), small intestine (n = 17), heart (n = 9), lung (n = 9) and spleen (n = 3).ConclusionsThe present study identified 2556 DEPs in six cat tissues on day 7 after infection by T. gondii PRU strain, and functional enrichment analyses showed that these DEPs were associated with various cellular and metabolic processes. These findings provide a solid base for further in-depth investigation of the complex proteotranscriptomic reprogramming that mediates the dynamic interplays between T. gondii and the different feline tissues.

Marker of schizophrenia with enduring negative symptoms

IntroductionThe relevance of this study is determined by the need to search for biological markers of schizophrenia. The detection and validation of such molecules can become the basis for the creation of additional paraclinical diagnostic methods or contribute to the creation of targets for individual pharmacotherapy, which is an important task of modern fundamental medicine.ObjectivesComparative proteomic analysis of serum in schizophrenic patients with positive and negative symptoms.MethodsThe study includes 10 healthy donors and 27 patients with schizophrenia. Samples preparation included: serum purification from major proteins via affinity chromatography, 1D-PAGE proteins separation, in-gel tryptic hydrolysis, LC-MS/MS mass-spectrometry (Orbitrap Q-exactive HF mass spectrometer, Agilent Technologies). Identification of proteins was carried out using Mascot software Ver. 2.1 («Matrix Science», USA). Proteins for quantitative analysis were selected in view of the DISGENET database. Quantitative LC-MS-SRM analysis of selected protein was performed on QQQ TSQ Vantage (Thermo Scientific) with labeled peptide standards.ResultsReceptor-interacting serine/threonine-protein kinase 1 was selected for quantitative assessment. Significant differences were revealed in the RIPK1 concentrations in the serum of schizophrenic patients with negative and positive symptoms (p=0.02). The serum concentration of RIPK1 in patients with negative symptoms is tenfold in patients with positive symptoms.ConclusionsReceptor-interacting serine/threonine-protein kinase 1 can be considered a biomarker of negative symptoms of schizophrenia based on a significant increase in serum concentration. Mass spectrometric analysis was carried out of the “Human Proteome” Core Facility of the Institute of Biomedical Chemistry Moscow. Support by Grant of RSF № 18-15-00053P.DisclosureNo significant relationships.

The Lysosome Origin of Biosilica Machinery in the Demospongiae Model Petrosia ficiformis (Poiret, 1789)

The silicification mechanism in sponges is a biologically controlled process where the complex and amazing shape of spicules is the result of the hierarchical assembly of silicon particles to form a composite structure with organic compounds, mainly constituted by proteins. In this work, using an integrated approach of transcriptomic and proteomic analysis, we describe the protein content of sponge spicules in the marine demosponge Petrosia ficiformis (Poiret, 1789). Proteins from spicules were obtained via an ammonium fluoride extraction procedure to remove the inorganic silica followed by SDS-PAGE electrophoresis. The resulting data of LC-MS/MS analysis of the extracted SDS-PAGE bands were then processed with the MASCOT software to search against a database consisting of transcripts and predicted proteins of P. ficiformis. The results revealed a very heterogeneous group of 21 proteins, including silicatein beta, different isoforms of cathepsins, proteins with strong homologies with enzymes like sulphatases, glycosidases, lipid-related proteins, phosphatases, and some others with unknown function. Most of the proteins found here have structures and domains attributable to lysosomes enzymes and for this reason it could be related to these cellular structures the evolutionary origin of the biosilica machinery in sponges.

Immunoproteomic analysis of the sporozoite antigens of Eimeria necatrix

Eimeria necatrix, an apicomplexan protozoa of the genus Eimeria, causes intestinal coccidiosis that can reduce growth performance of poultry and result in high mortality in older chickens. In this report, the whole sporozoite proteins of E.necatrix were studied by two-dimensional electrophoresis (2-DE) and Western blotting using hyper-immune chicken serum containing E.necatrix-specific antibodies. Approximately 680 protein spots for E.necatrix sporozoite were detected by 2-DE with silver staining, where 98 spots were cross-reacted with the E. necatrix-specific immune sera. Out of the 56 spots that were selected for MALDI-TOF-MS/MS analysis, 50 unique proteins were identified using the MASCOT software, 8 proteins were identified as known E.necatrix proteins and the rest were all putative proteins. These proteins have a wide range of known or predicted structures, cellular locations and functions, including proteins in category nuclear location & function, multifunctional- or multifunctional motifs-containing proteins, cellular transport and structure-related proteins, proteins of enzymatic activities, motor proteins-related, cell surface and organelle-related proteins. These new findings will enhance our understandings of parasite immunogenicity and immune evasion mechanisms of E. necatrix and facilitate the discovery phase of highly effective vaccine candidates.

Immunoproteomic analysis of fish ectoparasite, Argulus siamensis antigens.

An immunoproteomic approach was followed to identify immunoreactive antigens of fish ectoparasite, Argulus siamensis with rohu (Labeo rohita) immune sera for screening of potential vaccine candidates. The whole adult Argulus antigen was run in 2D electrophoresis with IEF in 7cm IPG strips of pH 4-7 and SDS-PAGE with 12% acrylamide concentration. Two parallel gels were run; one was stained with silver stain, and the other was Western blotted to nitrocellulose paper (NCP) and reacted with rohu anti-A siamensis sera. Fourteen protein spots corresponding to the spots developed in NCP were picked from the silver-stained gel and subjected to mass spectrometry in MALDI-TOF/TOF. The MS/MS spectra were analysed in MASCOT software with taxonomy 'other metazoa' and the proteins identified based on similarity with the proteins from heterologous species. The gene ontology analysis revealed a majority of proteins being involved in binding activity in 'molecular function' and belonging to metabolic processes in 'biologic process' categories. The possibility of these proteins as vaccine candidates against A siamensis is discussed in the paper. Three of the identified proteins namely, bromodomain-containing protein, anaphase-promoting complex subunit 5 and elongation factor-2 could possibly serve as vaccine candidates against argulosis in carps.

Peptidomic profiling of cerebrospinal fluid from patients with intracranial saccular aneurysms

Intracranial saccular aneurysms (ISA) represent 90%–95% of all intracranial aneurysm cases, characterizing abnormal pockets at arterial branch points. Ruptures lead to subarachnoid hemorrhages (SAH) and poor prognoses. We applied mass spectrometry-based peptidomics to investigate the peptidome of twelve cerebrospinal fluid (CSF) samples collected from eleven patients diagnosed with ISA. For peptide profile analyses, participants were classified into: 1) ruptured intracranial saccular aneurysms (RIA), 2) unruptured intracranial saccular aneurysms (UIA), and late-ruptured intracranial saccular aneurysms (LRIA). Altogether, a total of 2199 peptides were detected by both Mascot and Peaks software, from which 484 (22.0%) were unique peptides. All unique peptides presented conserved chains, domains, regions of protein modulation and/or post-translational modification sites related to human diseases. Gene Ontology (GO) analyses of peptide precursor proteins showed that 42% are involved in binding, 56% in cellular anatomical entities, and 39% in intercellular signaling molecules. Unique peptides identified in patients diagnosed with RIA have a larger molecular weight and a distinctive developmental process compared to UIA and LRIA (P ≤ 0.05). Continued investigations will allow the characterization of the biological and clinical significance of the peptides identified in the present study, as well as identify prototypes for peptide-based pharmacological therapies to treat ISA. Significance•Intracranial aneurysms (IAs) are arterial dilations that occur due to weakness in the media layer of the arterial wall. Due to lack of symptoms, IAs are usually found incidentally when the rupture occurs. The mechanism that causes vasospasms are not completely understood.•Here, twelve cerebrospinal fluid (CSF) samples collected from eleven patients diagnosed with IAs were investigated by mass spectrometry-based peptidomics.•2199 peptides were identified by both Mascot and Peaks software, from which 484 (22.0%) were unique peptides.•Even considering the relatively low number of patients enrolled in this seminal study, all unique peptides presented conserved chains, domains, regions of protein modulation and/or post-translational modification sites related to human diseases.•Gene Ontology (GO) analyses of peptide precursor proteins showed that 42% are involved in binding, 56% in cellular anatomical entities, and 39% in intercellular signaling molecules.•Unique peptides identified in patients diagnosed with RIA have a larger molecular weight and a distinctive developmental process compared to UIA and LRIA (P ≤ 0.05).•These results suggest peptides as novel ISA markers.•Continued investigations will allow the undoubted characterization of specific peptides that can be used as prototypes for peptide-based diagnostics and pharmacological therapies, to predict, prevent and/or treat ISA.

Systematic biological and proteomics strategies to explore the regulation mechanism of Shoutai Wan on recurrent spontaneous Abortion's biological network

Ethnopharmacological relevanceShoutai Wan (STW) is a classic herbal formula for the treatment of recurrent spontaneous abortion (RSA), and clinical studies have shown the effectiveness of STW on RSA. However, the molecular mechanism of STW treatment of RSA is still unclear. Methods(1) Animal experiments: The normal pregnancy model was established with CBA/J*BALB/C, and the RSA model was established by CBA/J*DBA/2. The RSA model CBA/J*DBA/2 pregnant mice were randomly divided into four groups (RSA model group, STW low, medium and high dose groups) according to the order of pregnancy, respectively. The drug administration starts from the first day of pregnancy to the 14th day of pregnancy. The embryo loss rate (ELR) of each group was calculated. (2) Proteomic analysis of decidual tissue: The total protein of decidual tissue of each group was isolated by solid phase pH gradient 2-DE technique. The differentially expressed protein spots were analyzed and identified by PDQuest images; the peptide quality fingerprinting (PMF) was obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Then, the proteins were identified by Mascot software searching, their functions were identified by bioinformatics strategy. (3) The expression of HSP27, α-enolase and Transferrin was detected by Western blotting and the expression of Annexin A2 and Transferrin was detected by immunohistochemistry. (4) The differential proteins and potential targets were analyzed by systematic biological strategy. Results(1) Compared with the normal group, the ELR in the RSA model group was significantly higher (P < 0.01). Compared with the model group, the ELR in the STW high, medium dose groups was lower (P < 0.01). (2) A 2-DE map of the decidual tissue of the RSA model group, normal pregnancy group, STW low, medium and high dose groups was established. Thirty proteins were identified. (3) The results of western blot showed that the expression of HSP27 and a-enolase in the RSA model group was higher than that in the normal group, and the expression of transferrin was lower (P < 0.01). Compared with the model group, the expression of HSP27 and a-enolase in STW high, medium dose groups was decreased (P < 0.01); Compared with the model group, the expression of Transferrin in the STW high dose group was increased (P < 0.01). (5) A lot of RSA treatment-related targets, biological processes and pathways were found after the systematic biological analysis. Conclusion(1) STW may reduce the ELR of the RSA mice. (2) The results of proteomics suggest that RSA is a complex process involving multiple proteins. STW can regulate the expression of various proteins in the decidual tissue of RSA mice, suggesting that it can act on multiple targets. (3) The results of western blotting of HSP27, a-enolase, transferrin were consistent with the results of proteomic analysis. (4) STW may achieve therapeutic effects by interfering with the targets, biological processes and signaling pathways discovered in this study.

Analysis of Proteomic Profile of Contrasting Phosphorus Responsive Rice Cultivars Grown under Phosphorus Deficiency

Phosphorus (P) deficiency is one of the major limiting factors for crop productivity. The yield of rice (Oryza sativa L.) is severely limited by phosphorus deficiency. An attempt has been made in this study to identify P deficiency responsive differentially expressed proteins of rice through analysis of leaf proteome of contrasting P-responsive rice cultivars under P deficiency conditions because genetic variability has been found in the rice cultivars for adaptive response to P deficiency and a controlled regulatory system is involved in the P deficiency adaptation response. Phosphorus-efficient (cv. Panvel) and P-inefficient (cv. Nagina 22) rice cultivars were hydroponically grown in the nutrient medium under control environmental conditions at low-P level (2.0 µM) and optimum-P level (320 µM) treatments. Expression patterns of the proteins of the leaves of both the cultivars were analyzed in 30-day-old plants. The identification of these proteins through mass spectrometry and MASCOT software (Matrix Science Inc., Boston, USA) revealed that these differentially expressed proteins were homologous to known functional proteins involved in energy metabolism, biosynthesis, photosynthesis, signaling, protein synthesis, protein folding, phospholipid metabolism, oxidative stress, transcription factors, and phosphorus metabolism. It has been observed that rice cultivars responded differently to low-P treatment through modification in protein expressions pattern to maintain the growth of the plants. Therefore, the expression patterns of proteins were different in both of the cultivars under low-P treatment. Higher potential of protein stability, stress tolerance, osmo-protection, and regulation of phosphorus uptake was observed in cv. Panvel than cv. Nagina 22. This study could help to unravel the complex regulatory process that is involved in adaptation to P deficiency in rice.

Optimization of Protein Extraction Method for 2DE Proteomics of Goat's Milk.

Two-dimensional electrophoretic (2DE)-based proteomics remains a powerful tool for allergenomic analysis of goat’s milk but requires effective extraction of proteins to accurately profile the overall causative allergens. However, there are several current issues with goat’s milk allergenomic analysis, and among these are the absence of established standardized extraction method for goat’s milk proteomes and the complexity of goat’s milk matrix that may hamper the efficacy of protein extraction. This study aimed to evaluate the efficacies of three different protein extraction methods, qualitatively and quantitatively, for the 2DE-proteomics, using milk from two commercial dairy goats in Malaysia, Saanen, and Jamnapari. Goat’s milk samples from both breeds were extracted by using three different methods: a milk dilution in urea/thiourea based buffer (Method A), a triphasic separation protocol in methanol/chloroform solution (Method B), and a dilution in sulfite-based buffer (Method C). The efficacies of the extraction methods were assessed further by performing the protein concentration assay and 1D and 2D SDS-PAGE profiling, as well as identifying proteins by MALDI-TOF/TOF MS/MS. The results showed that method A recovered the highest amount of proteins (72.68% for Saanen and 71.25% for Jamnapari) and produced the highest number of protein spots (199 ± 16.1 and 267 ± 10.6 total spots for Saanen and Jamnapari, respectively) with superior gel resolution and minimal streaking. Six milk protein spots from both breeds were identified based on the positive peptide mass fingerprinting matches with ruminant milk proteins from public databases, using the Mascot software. These results attest to the fitness of the optimized protein extraction protocol, method A, for 2DE proteomic and future allergenomic analysis of the goat’s milk.

P0666BRILLOUIN AND RAMAN SPECTROSCOPIES FOR NON-CONTACT ASSESSMENT OF MECHANO-CHEMICAL PROPORTIES OF URINARY PROTEINS: A PROOF OF CONCEPT STUDY

Abstract Background and Aims Proteinuria is a major marker of chronic kidney disease (CKD) severity, the predictor of renal function decline and cardiovascular mortality. The main pathway of CKD progressive is caused by tubular toxicity of filtering proteins, so more, the rapid development of renal failure is expected in those patients who have higher level of proteinuria. However, despite high level of proteinuria, paradoxically, some patients may have slow decline of renal function compared to those patients with lower level of urinary protein excretion. This may possibly due to their different mechanical (visco-elastic) and chemical properties, as well as the proteomics of excreting urinary proteins. Brillouin light scattering (BLS) and surface enhanced Raman scattering (SERS) spectroscopies are non-contact (laser optical-based) techniques providing visco-elastic and chemical property information of probed human biofluids. We aimed to study and compare the visco-elastic and chemical properties of urinary proteins using BLS and SERS spectroscopies in nephrotic patient with high protein excretion and healthy subject. Method Two urine samples from nephrotic patient (proteinuria&amp;gt;3.5 g/24h) and healthy subject (undetectable proteinuria) investigated using BLS and SERS spectroscopies. Brillouin spectroscopy performed using Torus single longitudinal mode laser in conjunction with high contrast JRS Instruments 6-pass tandem Fabry-Perot interferometer coupled with confocal microscope. Using Horiba LabRAM HR Raman microscope with 785 nm laser excitation andx100 microscope objective the SERS spectroscopy was performed on urine samples in contact 60 nm gold nanoparticles placed on gold film. Additionally, reversed-phase liquid chromatography-mass spectrometry (LC-MS) was conducted for profiling of urinary proteins LC-MS data was analysed by Mascot software using the SwissProt protein database. Results We found that elevated protein levels in urine alter the urine fluid elasticity sufficiently to be measurable by BLS spectroscopy, with urine from healthy and proteinuric subjects distinguishable to marked significance, which increases with sample concentration. BLS spectroscopy demonstrated that patients with High Protein Loss, characterized by stiffer urine (i.e. higher Brillouin shift and elastic modulus) and less viscous (i.e. less hypersound attenuation) compared to healthy subject. (Figure 1A). SERS measurements of two urine samples taken from patient with high protein loss in healthy subject (Figure 1B) showed the integrated Raman intensity ratio of 1338 cm-1 and 1002 cm-1 peaks is many times higher for the sample with high protein concentration vs sample with low protein concentration. Urinary protein sequencing and Mascot analysis (Table 1) demonstrated that proteinuric patient had different type of proteins with up to 187k Daltons of molecular weight. Conclusion In this proof of concept study, we demonstrated that laser-optical BLS and SERS techniques combined with LC-MS measurements revealed that human urine fluids of healthy and proteinuric subjects had strong variation in the visco-elastic and chemical properties coupled with various level of protein content.

Proteomics Analysis for Identification of Potential Cell Signaling Pathways and Protein Targets of Actions of Atractylodin and β-Eudesmol Against Cholangiocarcinoma.

Objective:The study aimed to identify potential cell signaling pathways and protein targets of actions of atractylodin and β-eudesmol in cholangiocarcinoma, the two active compounds isolated from Atracylodes lancea using proteomics approach. Method:The cholangiocarcinoma cell line, CL-6, was treated with each compound for 3 and 6 hours, and the proteins from both intra- and extracellular components were extracted. LC-MS/MS was applied following the separation of the extract proteins by SDS-PAGE and digestion with trypsin. Signaling pathways and protein expression were analyzed by MASCOT and STITCH software. Results:A total of 4,323 and 4,318 proteins were identified from intra- and extracellular components, respectively. Six and 4 intracellular proteins were linked with the signaling pathways (apoptosis, cell cycle control, and PI3K-AKT) of atractylodin and β-eudesmol, respectively. Four and 3 extracellular proteins were linked with the signaling pathways (NF-κB and PI3K-AKT) of atractylodin and β-eudesmol, respectively. Conclusion:In conclusion, a total of 17 proteins associated with four cell signaling pathways that could be potential molecular targets of anticholangiocarcinoma action of atractylodin and β-eudesmol were identified through the application of proteomics approach.

Proteome Profiling of the Mutagen-Induced Morphological and Yield Macro-Mutant Lines of Nigella sativa L.

In the present investigation, the leaf proteome profile of the macro-mutant lines of Nigella sativa L. was analyzed to identify the key proteins involved in the expression of traits associated with the morphology, seed yield, and content of thymoquinone. In our earlier study, the macro-mutants were generated with contrasting morphological traits and seed yields through induced mutagenesis, using ethyl methyl sulfonate, gamma rays, and combinations of both. Analysis of the leaf proteome of the control and macro-mutant lines of N. sativa showed that twenty-three proteins were differentially expressed. These differentially expressed proteins were sequenced through mass spectrometry and identified using the MASCOT software. On the basis of their function, these proteins were categorized into several groups. Most proteins were found in the categories of signal transduction (18%) and carbon metabolism (18%). A total of 13% of proteins belonged to the categories of energy and metabolism. Proteins in the categories of secondary plant metabolism, stress defense, cytoskeleton, and protein synthesis were also found. The polycomb group protein (FIE1), transcription factor (PRE1), and geranyl diphosphate synthase were notable proteins, in addition to some proteins of signal transduction and carbon metabolism. Expression patterns of the differentially expressed proteins were also studied at the transcript level by using qRT-PCR. Transcriptomics analysis was consistent with the proteomics data. This study shows the changes that take place at the proteomic level through induced mutagenesis, as well as the involvement of some proteins in the expression traits associated with plant height, seed yield, and the thymoquinone content of N. sativa. The identified proteins might help elucidate the metabolic pathways involved in the expression of traits, including seed yield, and the active compounds of medicinal plants.

Purification and characterization of an isoflavones conjugate hydrolyzing β-glucosidase (ICHG) from Cyamopsis tetragonoloba (guar)

A β-glucosidase with high specific activity towards isoflavone glycosidic conjugates was purified from seeds of Guar (Cyamopsis tetragonoloba) by ammonium sulphate precipitation followed by size exclusion and ion exchange chromatography. The pH and temperature optima of the purified Isoflavones conjugate hydrolyzing β-glucosidase (ICHG) were found to be pH 4.5 and 37 °C, respectively. The enzyme was relatively stable at higher temperatures. Effect of different divalent metal ions was studied and it was found that Cobalt and Mercury ions completely inhibited the enzyme activity. Km and Vmax of the purified isoflavones conjugates hydrolyzing β-glucosidases (ICHG) was 0.86 mM and 6.6 IU/mg respectively. The enzyme was most likely a trimer (approximate Mr 150 kDa) with potential subunits of 50 kDa. The purified enzyme showed activity against isoflavone conjugate glycosides viz daidzin and genistin but was inactive towards other flavonoid conjugates. The product conversion was confirmed by HPTLC and HRMS analysis. The MALDI-TOF analysis of the ICHG showed a score greater than 78 with 20 matches in MASCOT software. The five resultant peptides obtained had highest similarity in sequence with β-glucosidase from Cicer arietinum. The β-glucosidase from the C. arietinum has also been reported to exhibit the isoflavone conjugate hydrolyzing properties thus confirming the nature of the enzyme purified from the Guar seeds.

Identification of protein patterns in bovine placenta at early-mid pregnancy - Pilot studies.

Placenta is a crucial tissue for an appropriate development of the fetus and the course of pregnancy. Its composition and structure change dynamically along pregnancy but the full pattern of these changes is not fully described in cows yet. The aim of the present study was to detect qualitative and quantitative protein profiles of bovine placenta during early-mid pregnancy at the time of placental formation. Placental tissues from healthy cows (n = 3) in early pregnancy (3-5 months) were collected at the slaughterhouse. Maternal and fetal parts were manually divided prior to homogenization. Further analysis was done in triplicates on the maternal and fetal parts separately and subjected to one-dimensional (1D) electrophoretic separation, followed by identification of peptide maps by nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS/MS). Proteins were identified by use of the MASCOT software with the SwissProt database. Proteomic analysis showed more than 4000 differentially expressed proteins in maternal and fetal parts of placenta. Each part expressed around 900 proteins, of which ca. 90 were common. The identified proteins were analyzed in accordance to molecular function and their participation in biological processes. The obtained results provide new insight into the knowledge about biochemical characteristics of placenta (new proteins) and serve for further studies on the possible markers of physiological/pathological pregnancy or function of placenta. Moreover, our data can be a good starting point for further studies on the processes underlying the attachment of placenta.

Proteomic changes in Mycobacterium tuberculosis H37Rv under hyperglycemic conditions favour its growth through altered expression of Tgs3(Rv3234c) and supportive proteins (Rv0547c, AcrA1 and Mpa)

Diabetes affects the presentation of tuberculosis including delayed clearance of the bacteria from host cells, however, the molecular changes which help survival of phagocytosed mycobacterium in the diabetic host are still not clear. The effect of in vitro high glucose concentrations on the proteome of the phagocytosed mycobacterium isolated from the human monocytic THP1 cell line derived macrophages has been investigated in the present study. Concurrent tuberculosis and hyperglycemia conditions were mimicked by growing M. tuberculosis infected THP1 cells under high glucose conditions. Phagocytosed bacilli were isolated 5 days post infection. Proteomics analysis of the isolated bacilli was done by two-dimensional gel electrophoresis followed by mass spectrometry. A total of 224 ± 18 protein spots were obtained out of which 10 were found to be differentially expressed under high glucose concentrations in comparison to normal glucose concentration. Further, identity of all the ten proteins namely Tgs3, Rv0547c, AcrA1, EsxU, Rv2219, Mpa, Rv2308, ORN, LucA, and Rv1414 was elucidated by peptide mass finger printing using Matrix-assisted laser desorption and ionization-mass spectrometry (MALDI/MS) assisted with MASCOT software. Though Tgs3, Rv0547c, AcrA1 and Mpa proteins have been demonstrated to play a major role in lipid metabolism under nitric oxide stress conditions, the functional role of rest of the differentially expressed proteins remains to be elucidated. Under hyperglycemic conditions in the host cells, differential expression of these proteins might help in the better survival of mycobacteria and can further act as suitable targets to design novel drugs for more effective therapy for comorbid tuberculosis and diabetes.

Exploration of Mechanism for Meisoindigo-Inducing K562 Cell Apoptosis by Using Quantitative Proteomic Analysis

To screen the differentially expressed proteins at the early stage of K562 cells treated with meisoindigo by using tandem mass tags (TMT)-based proteomics technology, and to explore the mechanism for meisoindigo-inducing apoptosis. The half inhibitory concentration (IC50) of mesoindigo on K562 cells was determined by CCK8. The flow cytometry was used to assay the apoptosis of K562 cells treated by meisoindigo or DMSO. Total proteins were extracted from the cells treated with 0.2% DMSO (control) or 20 μmol/L meisoindigo (Test) for 2 hours. Then, the TMT-labeling HPLC-MS/MS was used to identify and quantify the peptides and their abundance, all the tests were repeated for 3 times. The Mascot software was used to identify the proteins; the GO annotations, enrichment and cluster analysis were used to analyze the differentially expressed proteins. Meisoindigo-induced K562 cell apoptosis in a dose-dependent manner (r=0.98), 5 544 proteins were identified, 4792 of which were quantified. The protein with expression difference>1.5-folds in Test group accoanted for 8, out of which the expression of 4 proteins were up-regulated and 4 were down-regulated. The differentially expressed proteins mainly associated with reactive oxygen species (ROS). Several proteins including DDIT4 were found to have dramatic changes in the early stage of K562 cells treated with meisoindigo by using quantitative proteomics technology. The ROS metabolic process may play important roles in meisoindigo-inducing apoptosis of K562 cells.

Differential proteomics analysis of bile between gangrenous cholecystitis and chronic cholecystitis

To establish human biliary protein expression profiles of gangrenous cholecystitis, chronic cholecystitis, and to discover differently expressed proteins for gangrenous cholecystitis by comparative proteomics, we gathered human gallbladder bile samples from gangrenous cholecystitis and chronic cholecystitis patients, respectively After removing the bile salts and lipid peptide fragments were identified by the iTRAQ-coupled LC-MS/MS technology,then identified in SwissProt with Mascot software. A total of 2251 proteins from chronic cholecystitis patients and 2180 proteins from gangrenous cholecystitis patients were identified. A total of 575 differential proteins were found between gangrenous cholecystitis and chronic cholecystitis, 159 proteins were over-expressed and 416 proteins were under-expressed in gangrenous cholecystitis. By bio-informatics analysis, in gangrenous cholecystitis, cell death, necrosis,immune response of neutrophils, apoptosis and degranulation of cells were activated; while cell survival, fatty acid metabolism, transport of molecular and proliferation of cells were inhibited, which might reflect the de-compensatory phase. Pathway analysis showed acute phase proteins were changed, indicating the role of the inflammatory response in the pathogenesis of gangrenous cholecystitis. Six acute phase proteins were found up-regulated,implying a close linkage to gangrenous gallbladder. Our study could be applicable in the biomarker discovery of gangrenous cholecystitis.