Proteome analysis of hairy root from Panax ginseng C. A. Meyer using peptide fingerprinting, internal sequencing and expressed sequence tag data

Abstract

As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS), respectively. More than 300 protein spots were detected on silver stained two-dimensional (2-D) gels using pH 3-10, 4-7, and 4.5-5.5 gradients. Major protein spots (159) were analyzed by peptide fingerprinting or de novo sequencing and the functions of 91 of these proteins were identified. Protein identification was achieved using the expressed sequence tag (EST) database from Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-TOF MS alone was insufficient for protein identification because of the lack of a genome database for Panax ginseng. Only 17 of the 159 protein spots were verified by peptide mass fingerprinting using MALDI-TOF MS whereas 87 out of 102 protein spots, which included 13 of the 17 proteins identified by MALDI-TOF MS, were identified by internal amino acid sequencing using tandem mass spectrometry analysis by ESI Q-TOF MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that a combination of internal sequencing and EST data analysis was an efficient identification method for proteome analysis of plants having incomplete genome data like ginseng. The 2-D patterns of the main root and leaves of Panax ginseng differed from that of the cultured hairy root, suggesting that some proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax ginseng.

Rooteomics: The Challenge of Discovering Plant Defense-Related Proteins in Roots

“…In this sense, a common approach for protein extraction has been to homogenize the plant material in the presence of liquid nitrogen, precipitate the protein content in a trichloroacetic acid/acetone solution and re-solubilize the pellet in a chaotropic agent [18]. Due to its simplicity, it remains the preferred protocol of total protein extraction from root tissue, as demonstrated by recent proteomic studies of wheat [19], maize [20], Medicago trunculata [21], rice [22] and ginseng [23] roots.…”

An Extraction Method Suitable for Two-Dimensional Electrophoresis of Low-abundant Proteins from Ginseng Roots

“…Dried samples were resolved in four different lysis buffers, respectively [16][17][18][19][20]. The common components they contained were 2 % CHAPS, 1 % PMSF, and 1 % protease inhibitor; and the differences were lysis buffers I: 8 M urea; Lysis buffers II: 7 M urea, 2 M thiourea; Lysis buffers III: 6 M urea, 2 M thiourea; Lysis buffers IV: 5 M urea, 2 M thiourea.…”

Overexpression of PgSQS1 Increases Ginsenoside Production and Negatively Affects Ginseng Growth Rate in Panax ginseng

“…Although more than 80 different skeletal types of triterpenes are known to occur naturally, the dammarane-type triterpenes are found only in a few species as major compounds, e.g., P. ginseng [2] and Gynostemma pentaphyllum (jiaogulan) [3]. Recent research including large-scale gene analysis [4][5] and proteome analysis [6][7] has shown that P. ginseng is one of the most suitable systems for studying the biosynthesis of dammarane-type triterpene saponins. The isoprenoid biosynthetic pathway plays an important role in plant metabolism.…”