Optimization of Protein Isolation and Label-Free Quantitative Proteomic Analysis in Four Different Tissues of Korean Ginseng

Abstract

Korean ginseng is one of the most valuable medicinal plants worldwide. However, our understanding of ginseng proteomics is largely limited due to difficulties in the extraction and resolution of ginseng proteins because of the presence of natural contaminants such as polysaccharides, phenols, and glycosides. Here, we compared four different protein extraction methods, namely, TCA/acetone, TCA/acetone–MeOH/chloroform, phenol–TCA/acetone, and phenol–MeOH/chloroform methods. The TCA/acetone–MeOH/chloroform method displayed the highest extraction efficiency, and thus it was used for the comparative proteome profiling of leaf, root, shoot, and fruit by a label-free quantitative proteomics approach. This approach led to the identification of 2604 significantly modulated proteins among four tissues. We could pinpoint differential pathways and proteins associated with ginsenoside biosynthesis, including the methylerythritol 4–phosphate (MEP) pathway, the mevalonate (MVA) pathway, UDP-glycosyltransferases (UGTs), and oxidoreductases (CYP450s). The current study reports an efficient and reproducible method for the isolation of proteins from a wide range of ginseng tissues and provides a detailed organ-based proteome map and a more comprehensive view of enzymatic alterations in ginsenoside biosynthesis.

Proteomic Analysis Reveals a Critical Role of the Glycosyl Hydrolase 17 Protein in Panax ginseng Leaves under Salt Stress

“…The total leaf proteins were isolated, as previously reported [ 65 ]. Briefly, 1 g of fine ground ginseng leaf powder was homogenized with 5 mL of ice-cold Tris-Mg/NP-40 extraction buffer (0.5 M Tris–HCl, pH 8.3; 2% ( v / v ) Nonidet P-40 (NP-40); 20 mM MgCl 2 ; 0.07% ( v / v ) β –mercaptoethanol) and centrifuged at 12,000 rpm for 10 min at 4 °C.…”

“…After alkylation, the trypsin solution, dissolved in 50 mM TEAB containing 5% acetonitrile (ACN) (enzyme to substrate ratio ( w / w ) of 1:50), was added and incubated at 37 °C overnight. The concentration of obtained peptides was measured using the Pierce Quantitative Fluorometric Peptide Assay kit (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s instructions [ 65 ]. The digested peptides were subsequently applied to desalting HLB OASIS columns and basic pH reversed-phase (BPRP) fractionation using an in-house developed stage tip, respectively, as reported elsewhere [ 68 ].…”

Advances in Discoveries of Plant Phytochemicals

“…Among the differentially expressed proteins between leaves and rhizomes of soft windflower (Anemone flaccida), most proteins involved in the metabolic pathway of triterpenoid saponins biosynthesis were upregulated in rhizomes (Zhan et al, 2016). Comparative proteome analysis of the leaves, roots, shoots and fruits of Korean Ginseng by label-free quantitative proteomics identified that 67 out of 1,179 differentially regulated proteins were associated with ginsenoside biosynthesis pathways, including MEP pathway, MVA pathway, UDP-glycosyltransferase and oxidoreductase (Van Nguyen et al, 2021).…”

Integration of high-throughput omics technologies in medicinal plant research: The new era of natural drug discovery

“…Among the differentially expressed proteins between leaves and rhizomes of soft windflower ( Anemone flaccida ), most proteins involved in the metabolic pathway of triterpenoid saponins biosynthesis were upregulated in rhizomes ( Zhan et al., 2016 ). Comparative proteome analysis of the leaves, roots, shoots and fruits of Korean Ginseng by label-free quantitative proteomics identified that 67 out of 1,179 differentially regulated proteins were associated with ginsenoside biosynthesis pathways, including MEP pathway, MVA pathway, UDP-glycosyltransferase and oxidoreductase ( Van Nguyen et al., 2021 ).…”