Project description:Heart tissue was lysed by sonication in 5% SDS (w/v) in 50 mM TEAB (pH 8.5) 450 microg of each sample was reduced with 10 mM DTT at 80 degC for 10 min and alkylated with 25 mM iodoacetamide in the dark for 30 min at room temperature. SDS was removed, and samples were digested with 20 microg of Sequencing Grade Modified Trypsin (Promega) using an S-trap mini device (Protifi) at 47 degC for 2 h. Lyophilized peptides were reconstituted in 100 microl of 200 mM TEAB buffer and labeled with 41 TMT10 reagents (lot# UL292365). After 2 h, reactions were quenched with hydroxylamine and combined. TMT-labeled peptides were fractionated by high pH reversed-phase (HPRP) chromatography. Briefly, peptides were reconstituted in 400 microl of 20 mM ammonium formate, and 125 microl was fractionated using a 2.1 mm x 5 cm BEH C18 column (Waters) and Waters ACQUITY iClass UPLC. Separations utilized a flow rate of 0.4 ml/min and column temperature of 55 degC, and mobile phases consisted of 20 mM ammonium formate, pH 10 (MPA) and neat MeCN (MPB). Separations used a gradient as follows: 0 min, 3% B; 3 min, 7% B; 50 min, 50% B, 51 min, 90% B; 55 min, 90% B; 56 min, 3% B; 60 min, 3% B. Forty-eight equal fractions were collected over 52 minutes into a 96-well plate containing 10 microl of 20% TFA per well. This analysis was repeated 2 times total for fractionation of 2 mg peptides. The first 3 fractions of each injection were excluded, and the remaining samples were re-concatenated into 12 fractions. Approximately 5 microg of each fraction was separately aliquoted for analysis of the unenriched proteome, and samples were lyophilized. Phosphopeptide enrichment used 200 microg of each of the 12 HPRP fractions was reconstituted in 80% MeCN/1% TFA containing 1M glycolic acid (buffer A), phosphopeptides were enriched using GL Sciences p10 TiO tips. After loading, tips were washed twice with buffer A followed by 2 times with 80% MeCN/1% TFA before elution with 20% MeCN/5% aqueous ammonia. After addition of neat formic acid, samples were lyophilized. Finally, peptides were desalted using C18 Stage Tips, lyophilized and reconstituted in 12 microl of 10 mM citrate in 1% TFA/2% MeCN. The unenriched fractions were reconstituted in 10 microl of 1% TFA/2% MeCN. 1D-LC-MS/MS was performed on 4.5 microl of each of the phospho-enriched fractions or on 0.75 microg of unenriched fractions. Samples were analyzed using a nanoACQUITY UPLC system (Waters) coupled to a coupled to a Exploris 480 high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source and FAIMS Pro Interface. Samples were first trapped on a Symmetry C18 180 microm x 20 mm trapping column (5 microl/min at 99.9/0.1 v/v H2O/MeCN) followed by an analytical separation using a 1.7 microm Acquity HSS T3 C18 75 microm x 250 mm column (Waters) with a 90 min gradient of 5 to 30% MeCN with 0.1% formic acid at a flow rate of 400 nl/min and column temperature of 55 degC. Data collection on was performed in data-dependent acquisition (DDA) mode with 3 FAIMS compensation voltages (-40, -60 and -80). Each CV used a 60,000 resolution full MS scan from m/z 375 to 1600 with a normalized AGC target of 300%, peptide monoisotopic peak determination, an intensity threshold of 5E3 ions, precursor fit of 70% with 0.7 m/z fit window, charge state of 2-5 and 40 s dynamic exclusion. MS/MS used a top6 method with 30,000 resolution and TurboTMT enabled, an isolation width of 0.7 m/z, NCE of 36, AGC target of 300% and maximum IT of 120 ms. Advanced precursor determination was enabled. The analysis of unenriched samples used a similar method except that a 1 s total scan time was used for each CV and MS/MS used an auto IT.

Project description:Background: Cyanobacteria are ecologically significant prokaryotes that can be found in heavy metals contaminated environments. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been extensively considered in cyanobacteria. Recently, most studies have been focused on different habitats using microalgae leads to a remarkable reduction of an array of organic and inorganic nutrients, but what takes place in the extracellular environment when cells are exposed to external supplementation with heavy metals remains largely unknown. Methods: Here, extracellular polymeric substances (EPS) production in strains Nostoc sp. N27P72 and Nostoc sp. FB71 was isolated from different habitats and thenthe results were compared and reported . Result: Cultures of both strains, supplemented separately with either glucose, sucrose, lactose, or maltose showed that production of EPS and cell dry weight were boosted by maltose supplementation. The production of EPS (9.1 ± 0.05 μg/ml) and increase in cell dry weight (1.01 ± 0.06 g/l) were comparatively high in Nostoc sp. N27P72 which was isolated from lime stones.The cultures were evaluated for their ability to remove Cu (II), Cr (III), and Ni (II) in culture media with and without maltose. The crude EPS showed metal adsorption capacity assuming the order Ni (II)> Cu (II)> Cr (III) from the metal-binding experiments .Nickel was preferentially biosorbed with a maximal uptake of 188.8 ± 0.14 mg (g cell dry wt) -1 crude EPS. We found that using maltose as a carbon source can increase the production of EPS, protein, and carbohydrates content and it could be a significant reason for the high ability of metal absorbance. FT-IR spectroscopy revealed that the treatment with Ni can change the functional groups and glycoside linkages in both strains. Results of Gas Chromatography-Mass Spectrometry (GC–MS) were used to determine the biochemical composition of Nostoc sp. N27P72, showed that strong Ni (II) removal capability could be associated with the high silicon containing heterocyclic compound and aromatic diacid compounds content. Conclusion: The results of this studyindicatede that strains Nostoc sp. N27P72 can be a good candidate for the commercial production of EPS and might be utilized in bioremediation field as an alternative to synthetic and abiotic flocculants.

Project description:Background: Cyanobacteria are ecologically significant prokaryotes that can be found in heavy metals contaminated environments. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been extensively considered in cyanobacteria. Recently, most studies have been focused on different habitats using microalgae leads to a remarkable reduction of an array of organic and inorganic nutrients, but what takes place in the extracellular environment when cells are exposed to external supplementation with heavy metals remains largely unknown. Methods: Here, extracellular polymeric substances (EPS) production in strains Nostoc sp. N27P72 and Nostoc sp. FB71 was isolated from different habitats and thenthe results were compared and reported . Result: Cultures of both strains, supplemented separately with either glucose, sucrose, lactose, or maltose showed that production of EPS and cell dry weight were boosted by maltose supplementation. The production of EPS (9.1 ± 0.05 μg/ml) and increase in cell dry weight (1.01 ± 0.06 g/l) were comparatively high in Nostoc sp. N27P72 which was isolated from lime stones.The cultures were evaluated for their ability to remove Cu (II), Cr (III), and Ni (II) in culture media with and without maltose. The crude EPS showed metal adsorption capacity assuming the order Ni (II)> Cu (II)> Cr (III) from the metal-binding experiments .Nickel was preferentially biosorbed with a maximal uptake of 188.8 ± 0.14 mg (g cell dry wt) -1 crude EPS. We found that using maltose as a carbon source can increase the production of EPS, protein, and carbohydrates content and it could be a significant reason for the high ability of metal absorbance. FT-IR spectroscopy revealed that the treatment with Ni can change the functional groups and glycoside linkages in both strains. Results of Gas Chromatography-Mass Spectrometry (GC–MS) were used to determine the biochemical composition of Nostoc sp. N27P72, showed that strong Ni (II) removal capability could be associated with the high silicon containing heterocyclic compound and aromatic diacid compounds content.

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Branched-chain alpha-ketoacids are preferentially reaminated and activate protein synthesis in the rat heart

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