Protein Extraction From Cowpea Tissues for 2-D Gel Electrophoresis and MS Analysis

Vasconcelos, None Emanuel; Nogueira, Fábio César Sousa; Abreu, Edith; Gonçalves, E. F.; Souza, P.; Campos, Francisco

doi:10.1365/s10337-005-0637-1

Cited by 31 publications

(20 citation statements)

References 14 publications

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“…2-DE gels and image analysis were performed as described by Vasconcelos et al (2005). Briefly, approximately 200 lg of total protein was loaded onto a rehydrated 11-cm Immobiline DryStrip pH 3-10 or pH 4-10 (GE Healthcare, USA).…”

Section: Two-dimensional Gel Electrophoresis (2-de)mentioning

confidence: 99%

“…The differentially displayed protein spots were excised from the acrylamide gels, destained and digested with sequencing-grade modified trypsin (Promega, Madison, WI, USA), as described by Vasconcelos et al (2005). Matrix-assisted laser desorption ionization-time of flighttandem mass spectrometry (MALDI TOF-TOF-MS/MS) acquisition was performed in an ABI 4700 Proteomics Analyzer (Applied Biosystems) using 3,5 dimethoxy-4-hydroxycinnamic acid as the matrix.…”

Section: Mass Spectrometrymentioning

confidence: 99%

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Heat and phosphate starvation effects on the proteome, morphology and chemical composition of the biomining bacteria Acidithiobacillus ferrooxidans

Ribeiro

Maretto

Nogueira

et al. 2010

World J Microbiol Biotechnol

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Acidithiobacillus ferrooxidans is a Gram negative, acidophilic, chemolithoautotrophic bacterium that plays an important role in metal bioleaching. During bioleaching, the cells are subjected to changes in the growth temperature and nutrients starvation. The aim of this study was to gather information about the response of the A.ferrooxidans Brazilian strain LR to K2HPO4 starvation and heat stress through investigation of cellular morphology, chemical composition and differential proteome. The scanning electron microscopic results showed that under the tested stress conditions, A. ferrooxidans cells became elongated while the Fourier transform infrared spectroscopy (FT-IR) analysis showed alterations in the wavenumbers between 850 and 1,275 cm(-1), which are related to carbohydrates, phospholipids and phosphoproteins. These findings indicate that the bacterial cell surface is affected by the tested stress conditions. A proteomic analysis, using 2-DE and tandem mass spectrometry, enabled the identification of 44 differentially expressed protein spots, being 30 due to heat stress (40°C) and 14 due to K2HPO4 starvation. The identified proteins belonged to 11 different functional categories, including protein fate, energy metabolism and cellular processes. The upregulated proteins were mainly from protein fate and energy metabolism categories. The obtained results provide evidences that A. ferrooxidans LR responds to heat stress and K2HPO4 starvation by inducing alterations in cellular morphology and chemical composition of the cell surface. Also, the identification of several proteins involved in protein fate suggests that the bacteria cellular homesostasis was affected. In addition, the identification of proteins from different functional categories indicates that the A. ferrooxidans response to higher than optimal temperatures and phosphate starvation involves global changes in its physiology.

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Section: Two-dimensional Gel Electrophoresis (2-de)mentioning

confidence: 99%

Section: Mass Spectrometrymentioning

confidence: 99%

Heat and phosphate starvation effects on the proteome, morphology and chemical composition of the biomining bacteria Acidithiobacillus ferrooxidans

Ribeiro

Maretto

Nogueira

et al. 2010

World J Microbiol Biotechnol

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“…Samples (100 mg each) were subjected to protein extraction performed essentially as described by Vasconcelos et al. . Proteins (100 μg per sample) were subjected to in‐solution trypsin digestion as described by Pinheiro et al.…”

mentioning

confidence: 99%

“…After harvesting, EFNs were immediately frozen in liquid nitrogen, freeze-dried and ground to a fine powder. Samples (100 mg each) were subjected to protein extraction performed essentially as described by Vasconcelos et al [5]. Proteins (100 g per sample) were subjected to in-solution trypsin digestion as described by Pinheiro et al [6].…”

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confidence: 99%

Time-course proteome analysis of developing extrafloral nectaries of Ricinus communis

et al. 2016

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Floral and extrafloral nectaries are unique organs that secrete energy rich chemical components, but their contribution for nectar production is largely unknown. Here, we present the first comparative proteome dataset of four developmental stages of the extrafloral nectaries from castor plant (Ricinus communis), an important biofuel crop. Respectively, from stage I-IV, we identified 626, 613, 449 and 356 proteins, respectively, summing up 882 nonredundant proteins. Surprisingly, we identified two isoforms of the potent toxin ricin, indicating that ricin expression is not limited to seeds, but it may serve a general defense purpose for the castor plant. To date, this is the most complete dataset of proteins either from floral or extrafloral nectaries, thus contributing to lay the foundations for investigations on their ecological and evolutionary importance.

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“…3.2.1 Preparation of protein sample and determination of the protein content The total protein of three groups of seeds were extracted by TCA/ acetone precipitation method respectively (Agnieszka and Juan, 2013;Vasconcelos et al, 2005;Wu et al, 2014). The concentration of total protein was measured by Bradford method (1976), and the protein content of three samples was calculated respectively.…”

Section: Methodsmentioning

confidence: 99%

Screening and Expression Analysis of Temperature-responsing Proteins that Can Remove Low Temperature Stagnancy of Seeds Germination of <i>Lepidium apetalum</i>

Junjie¹,

Zhao²,

Li³

et al. 2016

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As a kind of ephemeral plant, Lepidium apetalum Willd. in northern XinJiang shows special responsible characteristics to temperature during the germination: low temperature stratification could improve germination rate and homogeneity of L.apetalum seeds. But it could not germinate and be at stagnation stage under 4°C , while this phenomenon could be removed by the treatment with 25°C for 45 minutes or longer time. The mechanism why the treatment by higher temperature for a short time could remove germination stagnation under low temperature is not clear yet. In this study, the total proteins difference of seeds with three treatments were analyzed by 2-DE, which the seeds were without stratification, stayed at germination stagnation stage under low temperature and removed stagnation with 25°C treatment. Screened proteins responsing to temperature which were related to remove germination stagnation under low temperature, and the corresponding genes expression were confirmed by qRT-PCR. The result showed that there was no obvious difference between the seeds without stratification and the seeds at germination stagnation stage by low temperature. But the proteins expression was quite different between the seeds treated with 25°C for 45 minutes to remove the germination stagnation and the other two treated samples. 37 distinct protein spots were found in the 2-DE map with 14 up-regulated spots, 23 down-regulated spots. Among them, 6 up-regulated proteins of 14 spots were identified by LC-MS/MS, CDC48E, HSP17.6 and PER12were further chosen to be confirmed expression analysis by qRT-PCR. The result showed that their relative expression levels were significantly higher in seeds removed stagnation than in seeds before breaking stagnation. In addition, their expression levels were obviously increased with the time prolonging treated by 25°C with the highest level with the dealt for 45-55min and then maintainng this level of expression. The result showed that the genes expression levels were positively correlated to the germination percentagetreated with 25°C from 30-60 minutes. All the above suggested that Cdc48E, Hsp17.6, and Per12 induced by 25°C might be related to the germination stagnation removing of the seeds of L.apetalum. This conclusion provides a new clue for the study on the signal response to temperature in plants.

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Protein Extraction From Cowpea Tissues for 2-D Gel Electrophoresis and MS Analysis

Cited by 31 publications

References 14 publications

Heat and phosphate starvation effects on the proteome, morphology and chemical composition of the biomining bacteria Acidithiobacillus ferrooxidans

Heat and phosphate starvation effects on the proteome, morphology and chemical composition of the biomining bacteria Acidithiobacillus ferrooxidans

Time-course proteome analysis of developing extrafloral nectaries of Ricinus communis

Screening and Expression Analysis of Temperature-responsing Proteins that Can Remove Low Temperature Stagnancy of Seeds Germination of <i>Lepidium apetalum</i>

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