scholarly journals An efficient protein extraction method applied to mangrove plant Kandelia obovata leaves for proteomic analysis

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Jiao Fei ◽  
You-Shao Wang ◽  
Hao Cheng ◽  
Yu-Bin Su
Keyword(s):  
Proteomic Analysis ◽  
Protein Extraction ◽  
Elongation Factor ◽  
Extraction Methods ◽  
Vital Role ◽  
Kandelia Obovata ◽  
Protein Patterns ◽  
Mangrove Plant ◽  
B Method ◽  
Flight Mass Spectrometry

Abstract Background Mangroves plants, an important wetland system in the intertidal shores, play a vital role in estuarine ecosystems. However, there is a lack of a very effective method for extracting protein from mangrove plants for proteomic analysis. Here, we evaluated the efficiency of three different protein extraction methods for proteomic analysis of total proteins obtained from mangrove plant Kandelia obovata leaves. Results The protein yield of the phenol-based (Phe-B) method (4.47 mg/g) was significantly higher than the yields of the traditional phenol (Phe) method (2.38 mg/g) and trichloroacetic acid-acetone (TCA-A) method (1.15 mg/g). The Phe-B method produced better two-dimensional electrophoresis (2-DE) protein patterns with high reproducibility regarding the number, abundance and coverage of protein spots. The 2-DE gels showed that 847, 650 and 213 unique protein spots were separated from the total K. obovata leaf proteins extracted by the Phe-B, Phe and TCA-A methods, respectively. Fourteen pairs of protein spots were randomly selected from 2-DE gels of Phe- and Phe-B- extracted proteins for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) technique, and the results of three pairs were consistent. Further, oxygen evolving enhancer protein and elongation factor Tu could be observed in the 2-DE gels of Phe and Phe-B methods, but could only be detected in the results of the Phe-B methods, showing that Phe-B method might be the optimized choice for proteomic analysis. Conclusion Our data provides an improved Phe-B method for protein extraction of K. obovata and other mangrove plant tissues which is rich in polysaccharides and polyphenols. This study might be expected to be used for proteomic analysis in other recalcitrant plants.

scholarly journals Comparison of Different Protein Extraction Methods for Gel-Based Proteomic Analysis of Ganoderma spp.

2016 ◽  
Vol 35 (2) ◽  
pp. 100-106 ◽  
Author(s):  
Jameel R. Al-Obaidi ◽  
Noor Baity Saidi ◽  
Siti Rokhiyah Ahmad Usuldin ◽  
Siti Nahdatul Isnaini Said Hussin ◽  
Noornabeela Md Yusoff ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Protein Extraction ◽  
Extraction Methods

scholarly journals Modified TCA/acetone precipitation of plant proteins for proteomic analysis

2018 ◽  
Author(s):  
Liangjie Niu ◽  
Hang Zhang ◽  
Zhaokun Wu ◽  
Yibo Wang ◽  
Hui Liu ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Protein Modification ◽  
Protein Extraction ◽  
Extended Period ◽  
Extraction Methods ◽  
Precipitation Method ◽  
Plant Proteins ◽  
Prolonged Incubation ◽  
Acetone Precipitation ◽  
Modified Method

AbstractProtein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.

The Protein Extraction from Longan Pulp (Dimocarpus longan Lour. cv. Daw) for Proteomic Analysis Using One-Dimensional Electrophoresis

2019 ◽  
Vol 886 ◽  
pp. 21-26 ◽  
Author(s):  
Achara Kleawkla ◽  
Ekawit Threenet ◽  
Wanlapha Khonkham ◽  
Winai Wiriyaalongkorn ◽  
Adisak Joomwong ◽  
...  
Keyword(s):  
Protein Expression ◽  
Proteomic Analysis ◽  
Protein Extraction ◽  
Protein Pattern ◽  
Elongation Factor ◽  
Fruit Growth ◽  
Physiological Disorder ◽  
One Dimensional ◽  
Dimocarpus Longan ◽  
Dimensional Electrophoresis

Three procedures for protein extraction in longan pulp had been applied to analyze protein pattern and quality of Longan pulp (Dimocarpus longan Lour. cv. Daw) during fruit growth to increase protein expression in proteomic analysis at Maejo university’s farm. There were data points to compare between normal and physiological disorder syndromes during fruit growth (5,10, 15, 20, 25 and 30 weeks, respectively) by one dimensional electrophoresis (1-D gel) technique in reducing condition. The first protein extraction, M1 (95% ethanol) showed obviously 15 protein bands which molecular weights were 14.97, 17.90, 18.30, 21.63, 28.54, 31, 33.96, 35.02, 42, 51.69, 65.69, 71.54, 88.02, 106.86 and 130 kDa, respectively. While M2 extraction (phenol-methanol/ammonium acetate) and M3 extraction (1.5 mM tris-HCl pH 8.0, 5 mM EDTA, 2% SDS) had low protein expression and no sharpness (13 and 12 protein bands, respectively). In different extraction conditions, therefore, M1 was a suitable method because of highest protein bands and obvious protein expression on longan pulp for proteomic analysis. Proteomic analysis of M1 extraction method was used in protein analysis by using LC-MS / MS techniques. It was found that the heat shock protein 83 (81.0 kDa), a family of proteins that was produced by cells in response to exposure on stressful conditions, the elongation factor 1-alpha (49.45 kDa), a selective regulator of translation, and the peroxidase 4 (39.74 kDa), a protein that is involved in the degeneration or aging of cells. These proteins exhibited a darker appearance of the protein bands at 30 weeks. Moreover, a partial glyceraldehyde-3-phosphate dehydrogenase (34.06 kDa), the protein involved in metabolic processes in glucose degradation, was also founded a darker appearance at 25 weeks and low appearance at 30 weeks of abnormal longan. However, higher proteomic techniques should be studied to confirm this biomarker protein in the further.

Protein Extraction Methods Compatible with Proteomic Analysis for the Cotton Seedling

Crop Science ◽  
2009 ◽  
Vol 49 (2) ◽  
pp. 395-402 ◽  
Author(s):  
Chengjian Xie ◽  
De Wang ◽  
Xingyong Yang
Keyword(s):  
Proteomic Analysis ◽  
Protein Extraction ◽  
Extraction Methods ◽  
Cotton Seedling

Optimized protein extraction methods for proteomic analysis ofRhizoctonia solani

Mycologia ◽  
2008 ◽  
Vol 100 (6) ◽  
pp. 867-875 ◽  
Author(s):  
Dilip K. Lakshman ◽  
Savithiry S. Natarajan ◽  
Sukla Lakshman ◽  
Wesley M. Garrett ◽  
Arun K. Dhar
Keyword(s):  
Proteomic Analysis ◽  
Protein Extraction ◽  
Extraction Methods

scholarly journals Comparative Proteomic Analysis Reveals the Regulatory Effects of H2S on Salt Tolerance of Mangrove Plant Kandelia obovata

2019 ◽  
Vol 21 (1) ◽  
pp. 118 ◽  
Author(s):  
Yi-Ling Liu ◽  
Zhi-Jun Shen ◽  
Martin Simon ◽  
Huan Li ◽  
Dong-Na Ma ◽  
...  
Keyword(s):  
Salt Tolerance ◽  
Proteomic Analysis ◽  
Carbon Fixation ◽  
Triosephosphate Isomerase ◽  
High Salinity ◽  
Chlorophyll Biosynthesis ◽  
Primary Metabolism ◽  
Kandelia Obovata ◽  
Mangrove Species ◽  
Mangrove Plant

As a dominant mangrove species, Kandelia obovata is distributed in an intertidal marsh with an active H2S release. Whether H2S participates in the salt tolerance of mangrove plants is still ambiguous, although increasing evidence has demonstrated that H2S functions in plant responses to multiple abiotic stresses. In this study, NaHS was used as an H2S donor to investigate the regulatory mechanism of H2S on the salt tolerance of K. obovata seedlings by using a combined physiological and proteomic analysis. The results showed that the reduction in photosynthesis (Pn) caused by 400 mM of NaCl was recovered by the addition of NaHS (200 μM). Furthermore, the application of H2S enhanced the quantum efficiency of photosystem II (PSII) and the membrane lipid stability, implying that H2S is beneficial to the survival of K. obovata seedlings under high salinity. We further identified 37 differentially expressed proteins by proteomic approaches under salinity and NaHS treatments. Among them, the proteins that are related to photosynthesis, primary metabolism, stress response and hormone biosynthesis were primarily enriched. The physiological and proteomic results highlighted that exogenous H2S up-regulated photosynthesis and energy metabolism to help K. obovata to cope with high salinity. Specifically, H2S increased photosynthetic electron transfer, chlorophyll biosynthesis and carbon fixation in K. obovata leaves under salt stress. Furthermore, the abundances of other proteins related to the metabolic pathway, such as antioxidation (ascorbic acid peroxidase (APX), copper/zinc superoxide dismutase (CSD2), and pancreatic and duodenal homeobox 1 (PDX1)), protein synthesis (heat-shock protein (HSP), chaperonin family protein (Cpn) 20), nitrogen metabolism (glutamine synthetase 1 and 2 (GS2), GS1:1), glycolysis (phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI)), and the ascorbate–glutathione (AsA–GSH) cycle were increased by H2S under high salinity. These findings provide new insights into the roles of H2S in the adaptations of the K. obovata mangrove plant to high salinity environments.

scholarly journals An Optimized Protein Extraction Method for Gel-Free Proteomic Analysis of Opuntia Ficus-Indica

Plants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 115
Author(s):  
Akiko Hashiguchi ◽  
Hisateru Yamaguchi ◽  
Keisuke Hitachi ◽  
Kazuo Watanabe
Keyword(s):  
Salt Stress ◽  
Proteomic Analysis ◽  
Extraction Method ◽  
Protein Extraction ◽  
High Stress ◽  
Ribulose Bisphosphate Carboxylase ◽  
Protein Patterns ◽  
Opuntia Ficus Indica ◽  
Bisphosphate Carboxylase ◽  
Opuntia Spp

Opuntia spp. is an economically important vegetable crop with high stress-tolerance and health benefits. However, proteomic analysis of the plant has been difficult due to the composition of its succulent cladodes; the abundant polysaccharides interfere with protein extraction. To facilitate proteomic analysis of this plant, we present a rapid and simple protein extraction method for Opuntia ficus-indica (L.) Miller. The optimized method produced highly reproducible protein patterns and was compatible with a gel-free quantitative workflow without the need for additional purification. We successfully analyzed the cladode mesocarp and exocarp tissues, resulting in the identification of 319 proteins. In addition, we used this method to examine the relative changes in the Opuntia proteome in response to salt stress to determine whether physiological changes could be captured. Qualified observations were obtained, revealing that salt stress increased phosphoenolpyruvate carboxylase abundance and decreased ribulose-bisphosphate carboxylase in young O. ficus-indica plants. These findings suggest that Crassulacean acid metabolism is promoted under salinity. This study highlights the efficacy of our optimized protein extraction method for elucidating the metabolic adaptations of Opuntia using gel-free proteomic analysis.

scholarly journals Comparison of methods for the extraction of proteins from root and leaf tissue of sugarcane (Saccharum spp.) for proteomic analysis

2020 ◽  
pp. 1221-1229
Author(s):  
Luciana da Silva Viana ◽  
Paulo Pedro da Silva ◽  
Velber Xavier Nascimento ◽  
Alessandro Riffel ◽  
Antônio Euzébio Goulart Sant’Ana
Keyword(s):  
Proteomic Analysis ◽  
Qualitative Data ◽  
Ammonium Acetate ◽  
Control Method ◽  
Protein Extraction ◽  
Extraction Methods ◽  
Cell Wall Polysaccharides ◽  
Total Proteins ◽  
Protein Determination ◽  
Leaves And Roots

The extraction of proteins from plants is a crucial procedure for successful protein determination such as purification, separation, and mass spectrometry. Protein extraction from plant tissues is generally difficult due to the presence of various molecules (cell wall, polysaccharides, and lipids) and interfering compounds. For this reason, the step of separation of proteins is a big challenge in obtaining good results in plant proteomic studies, notably from sugarcane. The current study assesses three extraction methods to prepare protein samples for proteomic analysis. Method 1 (control): TCA/acetone, method 2: TCA/acetone modified and Method 3: Phenol/SDS/ammonium acetate. Plants of cultivar RB92579 were grown in 10L pots under ideal humidity conditions in a greenhouse for 60 days. Samples collected on leaves +1 and roots were carried out using nitrogen and stored in an ultra-freezer at -80ºC for later use in proteome assays. For the tested methods, a comparison was made between the quantitative and qualitative data obtained from the tissue of sugarcane leaves and roots. According to the results obtained, methods 2 and 3 produced the best yield in the extraction of total proteins from the leaves and roots of sugarcane, when compared to (control) method 1 (TCA/acetone). This can be observed when comparing the quantitative and qualitative data obtained using the different extraction methods. By comparing methods 2 and 3, the latter showed a massive gain of extracted proteins much greater than the first method, mainly when the extraction of total proteins from the roots are compared. Similarly, the 2-DE gels run after using method 3 showed less background, compared to method 2. Another observation was the presence of different “spots” in the 2-DE gels between the samples extracted using methods 2 and 3. Method 3 (phenol / SDS / ammonium acetate) presented better results for extraction of proteins and in the 2-DE gels, with a greater number of total and specific “spots”, greater reproducibility and less background. This method could be utilized as the standard method for proteomic studies in sugarcane.

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