Crop proteomics: Practical method for high resolution of two-dimensional electrophoresis

Panax ginseng roots are well known for their medicinal properties and have been used in Korean and Chinese traditional medicines for 1000s of years. However, the medicinal value of P. ginseng fruits remain poorly characterized. In this study, we used an integrated biochemical, proteomics, and metabolomics approach to look into the medicinal properties of ginseng fruits. DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS [2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)] assays showed higher antioxidant activities in ginseng fruits than leaves or roots. Two-dimensional gel electrophoresis (2-DE) profiling of ginseng fruit proteins (cv. Cheongsun) showed more than 400 spots wherein a total of 81 protein spots were identified by mass spectrometry using NCBInr, UniRef, and an in-house developed RNAseq (59,251 protein sequences)-based databases. Gene ontology analysis showed that most of the identified proteins were related to the hydrolase (18%), oxidoreductase (16%), and ATP binding (15%) activities. Further, a comparative proteome analysis of four cultivars of ginseng fruits (cvs. Yunpoong, Gumpoong, Chunpoong, and Cheongsun) led to the identification of 22 differentially modulated protein spots. Using gas chromatography-time of flight mass spectrometry (GC-TOF MS), 66 metabolites including amino acids, sugars, organic acids, phenolic acids, phytosterols, tocopherols, and policosanols were identified and quantified. Some of these are well known medicinal compounds and were not previously identified in ginseng. Interestingly, the concentration of almost all metabolites was higher in the Chunpoong and Gumpoong cultivars. Parallel comparison of the four cultivars also revealed higher amounts of the medicinal metabolites in Chunpoong and Gumpoong cultivars. Taken together, our results demonstrate that ginseng fruits are a rich source of medicinal compounds with potential beneficial health effects.

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“…Total proteins from fruits of four ginseng cultivars were isolated as described previously ( Kim U.G. et al, 2012 ; Gupta et al, 2015a ).…”

Section: Methodsmentioning

confidence: 99%

“…Two-dimensional electrophoresis (2-DE) and gel-image analysis were performed as reported previously ( Kim U.G. et al, 2012 ).…”

Section: Methodsmentioning

confidence: 99%

An Integrated Biochemical, Proteomics, and Metabolomics Approach for Supporting Medicinal Value of Panax ginseng Fruits

et al. 2016

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“…In-gel protein digestion was carried out according to Kim et al (2012). Briefly, selected CBB-stained protein spots were excised, washed with 200 µl 50% (v/v) acetonitrile in 0.1 M NH 4 HCO 3 , and vacuum-dried.…”

Section: In-gel Protein Digestionmentioning

confidence: 99%

“…Two-dimensional gel electrophoresis (2-DGE) analysis was performed as previously described (Kim et al, 2012). Briefly, the rehydration solution for each immobilised pH gradient (IPG) gel consisted of 7 M urea, 2 M thiourea, 4% (v/v) 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate (CHAPS), 2 M dithiothreitol (DTT), and 0.5% (v/v) IPG buffer, pH 4 -7 (GE Healthcare, Waukesha, WI, USA).…”

Section: -Dge Analysismentioning

confidence: 99%

Omics-based biomarkers for the identification of six Korean cultivars of sweet potato (Ipomoea batatasL. Lam)

Lee

Kim

et al. 2013

The Journal of Horticultural Science and Biotechnology

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“…Homogenate was centrifuged at 12 000 × g for 15 min at 4°C. Supernatant was then subjected to phenol extraction followed by methanolic ammonium acetate precipitation, for the recovery of total proteins . Briefly, the supernatant was mixed with equal volume of water‐saturated phenol by vigorous vortexing at room temperature.…”

mentioning

confidence: 99%

Abundant storage protein depletion from tuber proteins using ethanol precipitation method: Suitability to proteomics study

et al. 2015

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High-abundance proteins (HAPs) hamper in-depth proteome study necessitating development of a HAPs depletion method. Here, we report a novel ethanol precipitation method (EPM) for HAPs depletion from total tuber proteins. Ethanol showed a dose-dependent effect on depletion of sporamin from sweet potato and patatin from potato tubers, respectively. The 50% ethanol was an optimal concentration. 2DE analysis of EPM-prepared sweet potato proteins also revealed enrichment of storage proteins (SPs) in ethanol supernatant (ES) resulting in detection of new low-abundance proteins in ethanol pellet (EP), compared to total fraction. The ES fraction showed even higher trypsin inhibitor activity than total proteins, further showing the efficacy of EPM in enrichment of sporamin in ES fraction. Application of this method was demonstrated for comparative proteomics of two sweet potato cultivars (Hwang-geum and Ho-bac) and purification of SP (sporamin) in its native form, as examples. Comparative proteomics identified many cultivar specific protein spots and selected spots were confidently assigned for their protein identity using MALDI-TOF-TOF analysis. Overall, the EPM is simple, reproducible, and economical for depletion of SPs and is suitable for downstream proteomics study. This study opens a door for its potential application to other tuber crops or fruits rich in carbohydrates.

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Crop proteomics: Practical method for high resolution of two-dimensional electrophoresis

Cited by 7 publications

References 17 publications

An Integrated Biochemical, Proteomics, and Metabolomics Approach for Supporting Medicinal Value of Panax ginseng Fruits

An Integrated Biochemical, Proteomics, and Metabolomics Approach for Supporting Medicinal Value of Panax ginseng Fruits

Omics-based biomarkers for the identification of six Korean cultivars of sweet potato (Ipomoea batatasL. Lam)

Abundant storage protein depletion from tuber proteins using ethanol precipitation method: Suitability to proteomics study

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