A simple, economical and reproducible protein extraction protocol for proteomics studies of soybean roots

Rodrigues, Elisete Pains; Batista, Jesiane Stefania da Silva; Huergo, Luciano F.; Hungria, Mariangela

doi:10.1590/s1415-47572012000200016

Cited by 28 publications

(15 citation statements)

References 15 publications

(26 reference statements)

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“…Whole-cell proteins of soybean roots were extracted, from both the inoculated and non-inoculated treatments, following the simplified method described by Rodrigues et al [71]. IPG strips (pH 4–7, 13 cm, GE Healthcare, Uppsala, Sweden) were rehydrated overnight with aliquots of 350 μg of solubilized proteins.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional analysis of genes involved in nodulation in soybean roots inoculated with Bradyrhizobium japonicumstrain CPAC 15

et al. 2013

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BackgroundBiological nitrogen fixation in root nodules is a process of great importance to crops of soybean [Glycine max (L.) Merr.], as it may provide the bulk of the plant’s needs for nitrogen. Legume nodulation involves several complex steps and, although studied for many decades, much remains to be understood.ResultsThis research aimed at analyzing the global expression of genes in soybean roots of a Brazilian cultivar (Conquista) inoculated with Bradyrhizobium japonicum CPAC 15, a strain broadly used in commercial inoculants in Brazil. To achieve this, we used the suppressive subtractive hybridization (SSH) technique combined with Illumina sequencing. The subtractive library (non-inoculated x inoculated) of soybean roots resulted in 3,210 differentially expressed transcripts at 10 days after inoculation were studied. The data were grouped according to the ontologies of the molecular functions and biological processes. Several classes of genes were confirmed as related to N2 fixation and others were reported for the first time.ConclusionsDuring nodule formation, a higher percentage of genes were related to primary metabolism, cell-wall modifications and the antioxidant defense system. Putative symbiotic functions were attributed to some of these genes for the first time.

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“…Although there are several sample preparation protocols available in the literature (Palma et al 2011, Rodrigues et al 2012, protocols specifically designed for tomato species (Solanum lycopersicum L.) are not well defined.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of protein extraction methods for enhanced proteomic analysis of tomato leaves and roots

Vilhena

Franco

Schmidt

et al. 2015

An. Acad. Bras. Ciênc.

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Proteomics is an outstanding area in science whose increasing application has advanced to distinct purposes. A crucial aspect to achieve a good proteome resolution is the establishment of a methodology that results in the best quality and wide range representation of total proteins. Another important aspect is that in many studies, limited amounts of tissue and total protein in the tissue to be studied are found, making difficult the analysis. In order to test different parameters, combinations using minimum amount of tissue with 4 protocols for protein extraction from tomato (Solanum lycopersicum L.) leaves and roots were evaluated with special attention to their capacity for removing interferents and achieving suitable resolution in bidimensional gel electrophoresis, as well as satisfactory protein yield. Evaluation of the extraction protocols revealed large protein yield differences obtained for each one. TCA/acetone was shown to be the most efficient protocol, which allowed detection of 211 spots for leaves and 336 for roots using 500 µg of leaf protein and 800 µg of root protein per gel.

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“…Because of the biochemical heterogeneity, large amounts of proteases and oxidative enzymes, and the presence of non-protein contaminants can disturb the subsequent steps in proteomic approaches (Boonmee et al, 2011;Witzel et al, 2011). Therefore, there is not a universal protocol for extraction or a solvent that can capture an entire proteome, and adjustments to previously described protocols are required (Prinsi et al, 2011;Chatterjee et al, 2012;Rodrigues et al, 2012a;Zhang et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Method for obtaining high-resolution proteomic analysis from pericarps of guarana

Souza¹,

Angelo²,

Nogueira³

et al. 2014

Genet. Mol. Res.

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ABSTRACT. Guarana has great agricultural potential and is largely used therapeutically and in the production of non-alcoholic energy drinks. Genomic and proteomic studies are crucial to identify proteins that play central roles in the maintenance and viability of fruits, as well as to identify proteins related to the main metabolic pathways. However, the success of any protein analysis starts with the protein extract preparation, which needs to offer an extract that is free of contaminants. This study aimed to evaluate different extraction methods to obtain high-quantity and high-quality extracts that are compatible with analysis by 2-dimensional electrophoresis and tandem mass spectrometry protein identification. Three different methods were tested: trichloroacetic acid (TCA)/acetone, sodium dodecyl sulfate (SDS)/phenol, and polyvinylpolypyrrolidone (PVPP)/SDS/phenol. The extract obtained from the TCA/acetone precipitation presented low solubility and contamination with lipids and carbohydrates. On the Protein extraction from Paullinia cupana other hand, the quality of the extract gradually improved after using phenol and PVPP/phenol, enabling a yield up to 2 mg/g macerated tissues and the detection of 457 spots by 2-dimensional electrophoresis. The effectiveness of the procedure used was validated by identification of 10 randomly selected proteins by mass spectrometry. The procedure described here can be a starting point for applications using tissues of other organs of guarana or tissues of species that are similar to guarana.

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“…Although a single-step process for protein extraction would be highly desirable, no unique sample preparation method can be used to 2-DE analysis (Dunn, 1999). Therefore, many researchers developed and optimized some efficient methods such as a phenol/SDS-based method (Wang et al, 2006; Rodrigues et al, 2012) and non-phenol-based methods (Guerreiro et al, 1997; Natera et al, 2000), to find a simple method that could be applied regularly to proteomics studies of symbiosis interactions.…”

Section: Introductionmentioning

confidence: 99%

Proteomic insights into intra- and intercellular plant-bacteria symbiotic association during root nodule formation

Salavati¹,

Shafeinia²,

Klubicová³

et al. 2013

Front. Plant Sci.

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Over the last several decades, there have been a large number of studies done on the all aspects of legumes and bacteria which participate in nitrogen-fixing symbiosis. The analysis of legume–bacteria interaction is not just a matter of numerical complexity in terms of variants of gene products that can arise from a single gene. Bacteria regulate their quorum-sensing genes to enhance their ability to induce conjugation of plasmids and symbiotic islands, and various protein secretion mechanisms; that can stimulate a collection of chain reactions including species-specific combinations of plant-secretion isoflavonoids, complicated calcium signaling pathways and autoregulation of nodulation mechanisms. Quorum-sensing systems are introduced by the intra- and intercellular organization of gene products lead to protein–protein interactions or targeting of proteins to specific cellular structures. In this study, an attempt has been made to review significant contributions related to nodule formation and development and their impacts on cell proteome for better understanding of plant–bacterium interaction mechanism at protein level. This review would not only provide new insights into the plant–bacteria symbiosis response mechanisms but would also highlights the importance of studying changes in protein abundance inside and outside of cells in response to symbiosis. Furthermore, the application to agriculture program of plant–bacteria interaction will be discussed.

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“…Furthermore, while interfering compounds are challenging in all plant tissues, they are particularly abundant in green (i.e., photosynthetically active) tissues . As a result, numerous protein extraction methods using various combinations of acetone, methanol, chloroform, phenol, detergents, and molecular weight cutoff filters have been evaluated on plant tissues . However, nearly all the evaluations of extraction methods have examined compatibility with 2‐DE rather than reverse phase LC, which is necessary for deep and quantitative proteome profiling.…”

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confidence: 99%

Assessment and Refinement of Sample Preparation Methods for Deep and Quantitative Plant Proteome Profiling

2018

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A major challenge in the field of proteomics is obtaining high-quality peptides for comprehensive proteome profiling by LC-MS. Here, evaluation and modification of a range of sample preparation methods using photosynthetically active Arabidopsis leaf tissue are done. It was found that inclusion of filter-aided sample preparation (FASP) based on filter digestion improves all protein extraction methods tested. Ultimately, a detergent-free urea-FASP approach that enables deep and robust quantification of leaf and root proteomes is shown. For example, from 4-day-old leaf tissue, up to 11 690 proteins were profiled from a single sample replicate. This method should be broadly applicable to researchers working with difficult to process plant samples.

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A simple, economical and reproducible protein extraction protocol for proteomics studies of soybean roots

Cited by 28 publications

References 15 publications

Transcriptional analysis of genes involved in nodulation in soybean roots inoculated with Bradyrhizobium japonicumstrain CPAC 15

Evaluation of protein extraction methods for enhanced proteomic analysis of tomato leaves and roots

Method for obtaining high-resolution proteomic analysis from pericarps of guarana

Proteomic insights into intra- and intercellular plant-bacteria symbiotic association during root nodule formation

Assessment and Refinement of Sample Preparation Methods for Deep and Quantitative Plant Proteome Profiling

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