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.

A Simple Protein Extraction Method for Proteomic Analysis of Diverse Biological Specimens

2014 ◽  
Vol 10 (4) ◽  
pp. 298-311 ◽  
Author(s):  
Panga Reddy ◽  
Aishwarya Rao ◽  
Darpan Malhotra ◽  
Samridhi Sharma ◽  
Ravinder Kumar ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Extraction Method ◽  
Protein Extraction

scholarly journals Comparative Proteomic Analysis of Coregulation of CIPK14 and WHIRLY1/3 Mediated Pale Yellowing of Leaves in Arabidopsis

2018 ◽  
Vol 19 (8) ◽  
pp. 2231 ◽  
Author(s):  
Zhe Guan ◽  
Wanzhen Wang ◽  
Xingle Yu ◽  
Wenfang Lin ◽  
Ying Miao
Keyword(s):  
Protein Metabolism ◽  
Proteomic Analysis ◽  
Photochemical Efficiency ◽  
Time Of Flight ◽  
Photochemical Quenching ◽  
Relative Distribution ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Ps Ii ◽  
Bisphosphate Carboxylase

Pale yellowing of leaf variegation is observed in the mutant Arabidopsis lines Calcineurin B-Like-Interacting Protein Kinase14 (CIPK14) overexpression (oeCIPK14) and double-knockout WHIRLY1/WHIRLY3 (why1/3). Further, the relative distribution of WHIRLY1 (WHY1) protein between plastids and the nucleus is affected by the phosphorylation of WHY1 by CIPK14. To elucidate the coregulation of CIPK14 and WHIRLY1/WHIRLY3-mediated pale yellowing of leaves, a differential proteomic analysis was conducted between the oeCIPK14 variegated (oeCIPK14-var) line, why1/3 variegated (why1/3-var) line, and wild type (WT). More than 800 protein spots were resolved on each gel, and 67 differentially abundant proteins (DAPs) were identified by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF-MS). Of these 67 proteins, 34 DAPs were in the oeCIPK14-var line and 33 DAPs were in the why1/3-var line compared to the WT. Five overlapping proteins were differentially expressed in both the oeCIPK14-var and why1/3-var lines: ATP-dependent Clp protease proteolytic subunit-related protein 3 (ClpR3), Ribulose bisphosphate carboxylase large chain (RBCL), Beta-amylase 3 (BAM3), Ribosome-recycling factor (RRF), and Ribulose bisphosphate carboxylase small chain (RBCS). Bioinformatics analysis showed that most of the DAPs are involved in photosynthesis, defense and antioxidation pathways, protein metabolism, amino acid metabolism, energy metabolism, malate biosynthesis, lipid metabolism, and transcription. Thus, in the why1/3-var and oeCIPK14-var lines, there was a decrease in the photosystem parameters, including the content of chlorophyll, the photochemical efficiency of photosystem (PS II) (Fv/Fm), and electron transport rates (ETRs), but there was an increase in non-photochemical quenching (NPQ). Both mutants showed high sensitivity to intense light. Based on the annotation of the DAPs from both why1/3-var and oeCIPK14-var lines, we conclude that the CIPK14 phosphorylation-mediated WHY1 deficiency in plastids is related to the impairment of protein metabolism, leading to chloroplast dysfunction.

A protein extraction method compatible with proteomic analysis for the euhalophyteSalicornia europaea

2007 ◽  
Vol 28 (21) ◽  
pp. 3976-3987 ◽  
Author(s):  
Xuchu Wang ◽  
Xiaofang Li ◽  
Xin Deng ◽  
Heping Han ◽  
Wuliang Shi ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Extraction Method ◽  
Protein Extraction

Separation and Identification of Proteins Related to Fruits Ripening in Mulberry (Morus alba)

2011 ◽  
Vol 175-176 ◽  
pp. 25-29
Author(s):  
Wen Qian Liu ◽  
Li Li Zhou ◽  
Mei Su ◽  
Xu Juan Chi ◽  
Jian Zhong Tan
Keyword(s):  
Protein Extraction ◽  
Physiological Mechanism ◽  
Physiological Role ◽  
Ribulose Bisphosphate Carboxylase ◽  
Related Protein ◽  
Ribulose Bisphosphate ◽  
Specific Expression ◽  
Bisphosphate Carboxylase ◽  
Ripe Stage ◽  
Mulberry Fruits

To elucidate the physiological mechanism of mulberry fruit ripening in protein level, differential proteome expression of mulberry fruits was analyzed by using 2-DE and mass spectrometry in different ripening stages, green ripe stage(G), half ripe stage(R) and pan ripe stage(P). A mulberry cultivator, “Da10” was used as experimental material. The results showed that separation of proteins with 2-DE were significantly improved by using phenol/SDS buffer for protein extraction. 441, 222, 328 protein spots were detected respectively in ripening stage G, R and P. Among them, differential expression of 31 proteins was more than 2-fold and 6 proteins were stage-specific expression. 8 differential proteins were identified by MALDI-TOF/TOF MS analysis and database search, which were photosynthesis related proteins (ribulose bisphosphate carboxylase/oxygenase activase and ribulose bisphosphate carboxylase small subunit), stress related protein (18kD winter accumulating protein), glucose metabolism related protein (cell wall invertase）and so on, suggesting that these proteins may play the specific physiological role in mulberry fruits ripening.

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.

152. Optimization of protein extraction method for proteomic analysis of vanilla apices subjected to cryoprotective treatments

Cryobiology ◽  
2009 ◽  
Vol 59 (3) ◽  
pp. 412-413
Author(s):  
S.E. Valdés-Rodrı´guez ◽  
M.T. González-Arnao ◽  
B. Jiménez-Francisco ◽  
B. Durán-Sánchez ◽  
A. Guerrero ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Extraction Method ◽  
Protein Extraction
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