Abstract
Identification and quantification of different metabolites under stress, especially protein, is a vital way to understand plant adaptation mechanism. We established an efficient protein extraction method from the tiny amount (100 mg) of root tips of non-model medicinal plant Hyoscyamus albus, using bead-beating cell disruption, TRIzol extraction, and sequential chemical protein solubilization. H. albus is very well known for biosynthesized of different secondary metabolites like hyoscyamine, tropane alkaloids and scopolamine. Our method is rational for sample preparation even in small-scale proteomics of recalcitrant tissue and allows proficient, reproducible and impurity-free protein extraction. This method allows high-quality 2DE in mini-gel format (25 µg of protein/gel) for hydrophilic and hydrophobic sub-proteomes and is compatible to high-sensitive matrix-assisted laser/desorption ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF) mass spectrometry (MS). A protocol using TRIzol is more effective and reproducible to sequential chemical extraction of both hydrophilic and hydrophobic membrane proteins. We also demonstrated cell disrupted together with dithiothreitol (DTT) and polyvinylpolypyrrolidone (PVPP) is more useful to prevent polymerization of the phenolic compound than commonly used added DTT and PVPP with TRIzol reagent. Despite the unavailability of genomic sequence database, the efficacy of the protocol was also confirmed by MS/MS ion searches.
 
 J Bangladesh Agril Univ 17(4): 430–436, 2019
Highlights
2-DE and mass spectrometry (MS)-based proteomics is a promising approach to identify enzymes and protein factors involved in various metabolisms, which are essential for plant adaptation under different environmental stress conditions
Xiong et al (2011) reported simultaneous isolation of DNA, RNA, and protein from different tissues of M. truncatula, suggesting that TRIzol protocol should be modified to optimize to this plant
We have shown that our established protocol using References
Summary
2-DE and MS-based proteomics is a promising approach to identify enzymes and protein factors involved in various metabolisms, which are essential for plant adaptation under different environmental stress conditions. Proteins isolated from plant tissues, especially recalcitrant medicinal plants, are generally tough to resolve by 2-DE and to apply downstream MS analysis due to the presence of abundant interfering materials, such as charged metabolites, phenolics, polysaccharides, oxidative enzymes, and proteases (Aghaei and Komatsu, 2013; Isaacson et al, 2006; Faurobert et al, 2007). A significant effort has been paid to improve the enriching, extracting and partitioning the membrane proteins for proteomic analysis. The analysis of plant proteins including membrane proteins, remains challenging, especially with respect to the sample preparation of recalcitrant non-model medicinal plant tissues. Lack of genomic sequence databases is another substantial impediment in medicinal plant proteomics since numbers of high-quality MS/MS
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