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Optimization of protein extraction and two-dimensional gel electrophoresis profiles for the identification of Cordyceps sinensis and other similar species.

ABSTRACT: Given that Chinese materia medica (CMM) is expensive and rare, people take tremendous risk to adulterate and falsify Cordyceps sinensis with counterfeit species with similar morphological features. It is thus essential to develop new methods to identify the authenticity of Cordyceps sinensis. It is hypothesized in this study that Cordyceps sinensis possesses certain protein biomarkers distinct from its counterfeits, which can be identified by proteomic technologies for authentication purposes. This is the first study that aims to optimize the conditions for extracting proteins from Cordyceps sinensis, a hybrid of fungal-animal CMM, and to compare the two-dimensional gel electrophoresis (2-DE) profiles between different Cordyceps species. Two different protein extraction buffer systems, namely, phenol/sodium dodecyl sulfate (SDS) buffer or lysis buffer, were evaluated, where the preparation using lysis buffer yielded better protein content. The results also showed that extraction with lysis buffer without pre- or post-washing of samples was the most effective protocol, with over 220% of protein yield and 819 protein spots detected on a 2-DE gel. Moreover, the results demonstrated that Cordyceps sinensis possesses protein biomarkers distinct from its counterfeits, and these biomarkers are not source- or origin-dependent, strongly supporting the feasibility of using identified biomarkers as indicators for authentication of Cordyceps species. The findings of this study warrant further investigations on the structural identification of protein biomarkers of Cordyceps species.

SUBMITTER: Chan RCH

PROVIDER: S-EPMC6105017 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Optimization of protein extraction and two-dimensional gel electrophoresis profiles for the identification of Cordyceps sinensis and other similar species.

Chan Roy Chi Ho RCH Lam Steven Sai Wo SSW Fong Fiona Long Yan FLY Chan Dominic Tak Wah DTW Lee Fred Wang Fat FWF Sze Eric Tung Po ETP

PloS one 20180822 8

Given that Chinese materia medica (CMM) is expensive and rare, people take tremendous risk to adulterate and falsify Cordyceps sinensis with counterfeit species with similar morphological features. It is thus essential to develop new methods to identify the authenticity of Cordyceps sinensis. It is hypothesized in this study that Cordyceps sinensis possesses certain protein biomarkers distinct from its counterfeits, which can be identified by proteomic technologies for authentication purposes. T ...[more]

PMID: 30133529

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Project description:BackgroundProteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF) that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE) is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly.ResultsThe proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit revealed clearer presentation of the isoforms and increased intensities of the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gel images as compared with untreated SF samples and SF samples treated with acetone.ConclusionsThe acetone precipitation method and the combined treatment effect of acetone and 2-DE Clean-Up Kit are not preferred in preparing SF samples for 2-DE analysis as both protein intensities and numbers decrease significantly. On the other hand, 2-D Clean-Up Kit treated SF samples revealed clearer isoforms and higher intensities for the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gels. As a result, it is recommended that SF samples should be treated with protein clean up products such as 2-D Clean-Up Kit first before conducting proteomic research in searching for the relevant biomarkers associated with knee osteoarthritis.

Dataset Information

Optimization of protein extraction and two-dimensional gel electrophoresis profiles for the identification of Cordyceps sinensis and other similar species.

Publications

Optimization of protein extraction and two-dimensional gel electrophoresis profiles for the identification of Cordyceps sinensis and other similar species.

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