Dataset Information

Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach.

ABSTRACT: Shotgun proteomics protocols are widely used for the identification and/or quantitation of proteins in complex biological samples. Described here is a shotgun proteomics protocol that can be used to identify the protein targets of biologically relevant ligands in complex protein mixtures. The protocol combines a quantitative proteomics platform with a covalent modification strategy, termed Stability of Proteins from Rates of Oxidation (SPROX), which utilizes the denaturant dependence of hydrogen peroxide-mediated oxidation of methionine side chains in proteins to assess the thermodynamic properties of proteins and protein-ligand complexes. The quantitative proteomics platform involves the use of isobaric mass tags and a methionine-containing peptide enhancement strategy. The protocol is evaluated in a ligand binding experiment designed to identify the proteins in a yeast cell lysate that bind the well-known enzyme cofactor, ?-nicotinamide adenine dinucleotide (NAD+). The protocol is also used to investigate the protein targets of resveratrol, a biologically active ligand with less well-understood protein targets. A known protein target of resveratrol, cytosolic aldehyde dehydrogenase, was identified in addition to six other potential new proteins targets including four that are associated with the protein translation machinery, which has previously been implicated as a target of resveratrol.

SUBMITTER: Dearmond PD

PROVIDER: S-EPMC3208786 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach.

Dearmond Patrick D PD Xu Ying Y Strickland Erin C EC Daniels Kyle G KG Fitzgerald Michael C MC

Journal of proteome research 20110928 11

Shotgun proteomics protocols are widely used for the identification and/or quantitation of proteins in complex biological samples. Described here is a shotgun proteomics protocol that can be used to identify the protein targets of biologically relevant ligands in complex protein mixtures. The protocol combines a quantitative proteomics platform with a covalent modification strategy, termed Stability of Proteins from Rates of Oxidation (SPROX), which utilizes the denaturant dependence of hydrogen ...[more]

PMID: 21905665

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Project description:With the widespread use of traditional medicine around the world, the safety and efficacy of traditional herbal patent medicine have become an increasing concern to the public. However, it is difficult to supervise the authenticity of herbal materials in mixed herbal products according to the current quality standards, especially for traditional herbal patent medicine, with a distinct variance in the dosage of herbal materials. This study utilized the shotgun metabarcoding approach to analyze the biological ingredients of Fuke Desheng Wan (FKDSW), which is an effective traditional herbal product for the treatment of dysmenorrhea. Six herbal materials were collected, and a lab-made mock FKDSW sample was produced to establish a method for the authentication assessment of biological ingredients in traditional herbal patent medicine based on shotgun metabarcoding. Furthermore, four commercial FKDSW samples were collected to verify the practicality of the shotgun metabarcoding approach. Then, a total of 52.16 Gb raw data for 174 million paired-end reads was generated using the Illumina NovaSeq sequencing platform. Meanwhile, 228, 23, and 14 operational taxonomic units (OTUs) were obtained for the ITS2, matK, and rbcL regions, respectively, after bioinformatic analysis. Moreover, no differences were evident between the assembly sequences obtained via shotgun metabarcoding and their corresponding reference sequences of the same species obtained via Sanger sequencing, except for part of the ITS2 and matK assembly sequences of Paeonia lactiflora Pall., Saussurea costus (Falc.) Lipsch. and Bupleurum chinense DC. with 1-6 different bases. The identification results showed that all six prescribed ingredients were successfully detected and that the non-authentic ingredient of Bupleuri Radix (Chaihu, Bupleurum chinense DC. or Bupleurum scorzonerifolium Willd.) was found in all the commercial samples, namely Bupleurum falcatum L. Here, 25 weed species representing 16 genera of ten families were detected. Moreover, 26 fungal genera belonging to 17 families were found in both lab-made and commercial FKDSW samples. This study demonstrated that the shotgun metabarcoding approach could overcome the biased PCR amplification and authenticate the biological ingredients of traditional herbal patent medicine with a distinct variance in the dosage of the herbal materials. Therefore, this provides an appropriate evaluation method for improving the safety and efficacy of traditional herbal patent medicine.

Dataset Information

Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach.

Publications

Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach.

Similar Datasets

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