ABSTRACT
Ginseng is a perennial herb of the genus Panax in the family Araliaceae as one of the most important traditional medicine. Genomic studies of ginseng assist in the systematic discovery of genes related to bioactive ginsenosides biosynthesis and resistance to stress, which are of great significance in the conservation of genetic resources and variety improvement. The transcriptome reflects the difference and consistency of gene expression, and transcriptomics studies of ginseng assist in screening ginseng differentially expressed genes to further explore the powerful gene source of ginseng. Protein is the ultimate bearer of ginseng life activities, and proteomic studies of ginseng assist in exploring the biosynthesis and regulation of secondary metabolites like ginsenosides and the molecular mechanism of ginseng adversity adaptation at the overall level. In this review, we summarize the current status of ginseng research in genomics, transcriptomics and proteomics, respectively. We also discuss and look forward to the development of ginseng genome allele mapping, ginseng spatiotemporal, single-cell transcriptome, as well as ginseng post-translational modification proteome. We hope that this review will contribute to the in-depth study of ginseng and provide a reference for future analysis of ginseng from a systems biology perspective.
Subject(s)
Ginsenosides , Panax , Panax/genetics , Proteomics , Gene Expression Profiling , Genome, Plant , Plant Roots/metabolismABSTRACT
Panax ginseng is a precious and ancient medicinal plant. The completion of its genome sequencing has laid the foundation for the study of proteome and peptidome. However, the high abundance of secondary metabolites in ginseng reduces the identification efficiency of proteins and peptides in mass spectrometry. In this report, strong cation exchange pretreatment was carried out to eliminate the interference of impurities. Based on the charge separation of proteolytic peptides and metabolites, the sensitivity of mass spectrometry detection was greatly improved. After pretreatment, 2322 and 2685 proteins were identified from the root and stem leaf extract. Further, the ginseng peptidome was analyzed based on this optimized strategy, where 970 and 653 endogenous peptides were identified from root and stem leaf extract, respectively. Functional analysis of proteins and endogenous peptides provided valuable information on the biological activities, metabolic processes, and ginsenoside biosynthesis pathways of ginseng.
Subject(s)
Ginsenosides , Panax , Panax/chemistry , Proteomics , Mass Spectrometry , Chromatography, Liquid , Ginsenosides/analysis , Plant Extracts/chemistry , Peptides/analysis , Plant Roots/chemistry , Chromatography, High Pressure LiquidABSTRACT
Protein tyrosine phosphorylation (pTyr) plays a prominent role in signal transduction and regulation in all eukaryotic cells. In conventional immunoaffinity purification (IP) methods, phosphotyrosine peptides are isolated from the digest of cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. However, low sensitivity, poor reproducibility, and high cost are universal concerns for IP approaches. In this study, we presented an antibody-free approach to identify phosphotyrosine peptides by using protein tyrosine phosphatase (PTP). It was found that most of the PTPs including PTP1B, TCPTP, and SHP1 can efficiently and selectively dephosphorylate phosphotyrosine peptides. We then designed a workflow by combining two Ti4+-IMAC-based phosphopeptide enrichment steps with PTP-catalyzed dephosphorylation for tyrosine phosphoproteomics analysis. This workflow was first validated by selective detection of phosphotyrosine peptides from semicomplex samples and then applied to analyze the tyrosine phosphoproteome of Jurkat T cells. Around 1000 putative former phosphotyrosine peptides were identified from less than 500 µg of cell lysate. The tyrosine phosphosites on the majority of these peptides could be unambiguously determined for over 70% of them possessing only one tyrosine residue. It was also found that the tyrosine sites identified by this method were highly complementary to those identified by the SH2 superbinder-based method. Therefore, the combination of Ti4+-IMAC enrichment with PTP dephosphorylation provides an alternative and cost-effective approach for tyrosine phosphoproteomics analysis.
Subject(s)
Proteomics , Tyrosine , Humans , Peptides/chemistry , Phosphorylation , Phosphotyrosine/chemistry , Protein Tyrosine Phosphatases , Proteome/analysis , Proteomics/methods , Reproducibility of Results , Tyrosine/chemistryABSTRACT
High-throughput drug discovery is highly dependent on the targets available to accelerate the process of candidates screening. Traditional chemical proteomics approaches for the screening of drug targets usually require the immobilization/modification of the drug molecules to pull down the interacting proteins. Recently, energetics-based proteomics methods provide an alternative way to study drug-protein interaction by using complex cell lysate directly without any modification of the drugs. In this study, we developed a novel energetics-based proteomics strategy, the solvent-induced protein precipitation (SIP) approach, to profile the interaction of drugs with their target proteins by using quantitative proteomics. The method is easy to use for any laboratory with the common chemical reagents of acetone, ethanol, and acetic acid. The SIP approach was able to identify the well-known protein targets of methotrexate, SNS-032, and a pan-kinase inhibitor of staurosporine in cell lysate. We further applied this approach to discover the off-targets of geldanamycin. Three known protein targets of the HSP90 family were successfully identified, and several potential off-targets including NADH dehydrogenase subunits NDUFV1 and NDUFAB1 were identified for the first time, and the NDUFV1 was validated by using Western blotting. In addition, this approach was capable of evaluating the affinity of the drug-target interaction. The data collectively proved that our approach provides a powerful platform for drug target discovery.
Subject(s)
HSP90 Heat-Shock Proteins/antagonists & inhibitors , Methotrexate/pharmacology , NADH Dehydrogenase/antagonists & inhibitors , Oxazoles/pharmacology , Proteomics , Staurosporine/pharmacology , Thiazoles/pharmacology , Acetic Acid/chemistry , Acetone/chemistry , Cells, Cultured , Drug Discovery , Drug Evaluation, Preclinical , Ethanol/chemistry , HEK293 Cells , HSP90 Heat-Shock Proteins/chemistry , HeLa Cells , High-Throughput Screening Assays , Humans , Methotrexate/chemistry , NADH Dehydrogenase/chemistry , NADH Dehydrogenase/metabolism , Oxazoles/chemistry , Solvents/chemistry , Staurosporine/chemistry , Thiazoles/chemistryABSTRACT
This study aimed to develop a practical ratiometric fluorescent probe for highly selective and sensitive detection of human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most important phase II enzymes. 4-Hydroxy-1,8-naphthalimide (HN) was selected as the fluorophore for this study because it possesses intramolecular charge transfer (ICT) feature and displays outstanding optical properties. A series of N-substituted derivatives with various hydrophobic, acidic and basic groups were designed and synthesized to evaluate the selectivity of HN derivatives toward UGT1A1. Our results demonstrated that the introduction of an acidic group to HN could significantly improve the selectivity of UGT1A1. Among the synthesized fluorescent probes, NCHN (N-3-carboxy propyl-4-hydroxy-1,8-naphthalimide) displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following UGT1A1-catalyzed glucuronidation. UGT1A1-catalyzed NCHN-4-O-glucuronidation generated a single fluorescent product with a high quantum yield (Φ=0.688) and brought remarkable changes in both color and fluorescence in comparison with the parental substrate. The newly developed probe has been successfully applied for sensitive measurements of UGT1A1 activities in human liver preparations, as well as for rapid screening of UGT1A1 modulators, using variable enzyme sources. Furthermore, its potential applications for live imaging of endogenous UGT1A1in cells have also been demonstrated.
Subject(s)
Enzyme Assays/methods , Fluorescent Dyes/chemistry , Glucuronosyltransferase/analysis , Microsomes, Liver/enzymology , Naphthalimides/chemistry , Biosensing Techniques/methods , Drug Evaluation, Preclinical/methods , Fluorescent Dyes/metabolism , Glucuronosyltransferase/antagonists & inhibitors , Glucuronosyltransferase/metabolism , Hep G2 Cells , Humans , Microscopy, Fluorescence/methods , Naphthalimides/metabolism , Optical Imaging/methodsABSTRACT
Resibufogenin (RB), one of the major active compounds of the traditional Chinese medicine Chansu, has displayed great potential as a chemotherapeutic agent in oncology. However, it is a digoxin-like compound that also exhibits extremely cardiotoxic effects. The present study aimed to characterize the metabolic behaviors of RB in humans as well as to evaluate the metabolic effects on its bioactivity and toxicity. The phase I metabolic profile in human liver microsomes was characterized systemically, and the major metabolite was identified as marinobufagenin (5ß-hydroxylresibufogenin, 5-HRB) by liquid chromatography-mass spectrometry and nuclear magnetic imaging techniques. Both cytochrome P450 (P450) reaction phenotyping and inhibition assays using P450-selective chemical inhibitors demonstrated that CYP3A4 was mainly involved in RB 5ß-hydroxylation with much higher selectivity than CYP3A5. Kinetic characterization demonstrated that RB 5ß-hydroxylation in both human liver microsomes and human recombinant CYP3A4 obeyed biphasic kinetics and displayed similar apparent kinetic parameters. Furthermore, 5-HRB could significantly induce cell growth inhibition and apoptosis in A549 and H1299 by facilitating apoptosome assembly and caspase activation. Meanwhile, 5-HRB displayed very weak cytotoxicity of human embryonic lung fibroblasts, and in mice there was a greater tolerance to acute toxicity. In summary, CYP3A4 dominantly mediated 5ß-hydroxylation and was found to be a major metabolic pathway of RB in the human liver, whereas its major metabolite (5-HRB) displayed better druglikeness than its parent compound RB. Our findings lay a solid foundation for RB metabolism studies in humans and encourage further research on the bioactive metabolite of RB.
Subject(s)
Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Bufanolides/metabolism , Bufanolides/pharmacology , Metabolic Detoxication, Phase I/physiology , Animals , Antineoplastic Agents/adverse effects , Bufanolides/adverse effects , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 Enzyme System/metabolism , Dogs , Guinea Pigs , Humans , Hydroxylation/physiology , Kinetics , Liver/metabolism , Macaca fascicularis , Male , Mice , Mice, Inbred ICR , Microsomes, Liver/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Peptides in urine are excreted by kidney from the blood and tissues, which are composed of a large amount of hormones, cytokines, regulatory factors and the metabolized fragments of proteins. The peptide distribution in urine will reflect the physiological and pathophysiological processes in body. In past, limited information was reported about the composition of the peptides in urine. One possible reason is that the peptides in urine are fairly low abundant and there are high concentrations of salts and organic metabolites in the urine. In this report, we extracted the peptides from human urine by highly ordered mesoporous silica particles with the pore size of 2 nm, which will exclude the high molecular weight proteins over 12 kDa. The extracted peptides were then separated into fractions according to their molecular weight by size exclusion chromatography. Each of the fractions was further analyzed by MALDI-TOF MS and µRPLC-MS/MS. Totally, 193 peptides were identified by two-dimensional SEC/µRPLC-MS/MS analysis. By analyzing the progenitor protein of the peptides; we found that two-thirds of the proteins differed from the reported urine proteome database, and the high abundant proteins in urine proteome were less detected in the urine peptidome. The developed extraction and separation methods were efficient for the profiling of the endogenous peptides in human urine. The peptidome in human urine was complementary to the human urinary proteome and may provide an emerging field for biomarker discovery.
Subject(s)
Peptides/urine , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Biomarkers/urine , Chromatography, Gel , Chromatography, Reverse-Phase , Humans , Nanoparticles , Peptides/isolation & purification , Porosity , Proteome/analysis , Silicon Dioxide/chemistryABSTRACT
A comprehensive two-dimensional HPLC system with an immobilized liposome chromatography (ILC) column in conjunction with an RP column (in tandem) was developed for the screening and analysis of the membrane-permeable compounds in a traditional Chinese medicine prescription Longdan Xiegan Decoction (LXD). More than 50 components in LXD were resolved using the developed separation system. Eight flavonoids and two iridoids were identified interacting with the ILC column; a system that mimics biomembranes. The results show that the developed comprehensive two-dimensional chromatography system can be used for identifying membrane permeable natural products in complex matrixes such as extracts of traditional Chinese medicine prescriptions.
Subject(s)
Chromatography, High Pressure Liquid/methods , Drugs, Chinese Herbal/chemistry , Flavonoids/analysis , Iridoids/analysis , Liposomes , Mass Spectrometry , Membranes, ArtificialABSTRACT
A comprehensive two-dimensional biochromatography method using a silica-bonded human serum albumin (HSA) column and a RP-HPLC column was developed for the biological fingerprinting analysis of bioactive components in a traditional Chinese medicine (TCM) prescription, Longdan Xiegan Decoction (LXD). The biochromatography with HSA-immobilized stationary phase was applied to study the interaction of multiple components in LXD with HSA in the first dimension, and fractions of HSA column were further separated by a silica monolithic ODS column (on-line)/an ODS column (off-line) coupled with a diode array detector and an atmospheric pressure chemical ionization mass spectrometer (APCI-MS). More than 100 compounds in LXD that interacted with the immobilized HSA were separated and analyzed. Among them 19 compounds were identified based on their retention values, UV spectra, molecular weights and mass spectra. The results show that the developed comprehensive two-dimensional biochromatography system reported here is capable of being used for biological fingerprinting analysis of natural products in complex matrices such as extracts of TCMs and their prescriptions.
Subject(s)
Chromatography, Liquid/methods , Drugs, Chinese Herbal/chemistry , Mass Spectrometry/methods , Molecular Weight , Spectrophotometry, UltravioletABSTRACT
A novel technique for removal of three-dimensional background drift in comprehensive two-dimensional (2D) liquid chromatography coupled with diode array detection (LCxLC-DAD) data is proposed. The basic idea is to perform trilinear decomposition on the instrumental response data, which is based on the alternating trilinear decomposition (ATLD) algorithm. In model construction, the background drift is modeled as one component or factor as well as the analytes of interest, hence, the drift is explicitly included into the calibration. The method involves performing trilinear decomposition on the raw data, then extracting the background component and subtracting this background data from the raw data, leaving the analytes' signal on a flat baseline. Simultaneous evaluation of three-dimensional background drift and true signals may improve the quality of the data. This method is applied to the determination and removal of three-dimensional background drifts in simulated multidimensional data as well as experimental comprehensive two-dimensional liquid chromatographic data. It is shown that this technique yield a good removal of background drift, without the need to perform a blank chromatographic run, and required no prior knowledge about the sample composition.
Subject(s)
Chromatography, Liquid/methods , Models, Theoretical , Algorithms , Calibration , Computer Simulation , Drugs, Chinese Herbal/analysis , Drugs, Chinese Herbal/chemistry , Ligusticum , Reproducibility of Results , Spectrophotometry, UltravioletABSTRACT
A stationary phase for high performance affinity chromatography with immobilization of DNA onto silica gel was prepared and characterized. The effect of the ionic strength, concentration of Mg2+, EDTA and CH3CN in the mobile phase on the retention of alkaloids were investigated. With this stationary phase, biological fingerprinting analysis of traditional Chinese medicines (TCMs) Coptis chinensis Franch and Rheum palmatum L. was performed with both one-dimensional (1-D) and two-dimensional (2-D) chromatography. The 1-D chromatography was performed with isocratic and gradient elution and 2-D chromatography was developed with immobilized DNA column combined with silica monolithic ODS column. It was found that 7 compounds in Coptis chinensis Franch including berberine, palmatine and jatrorrhizine, 14 compounds in Rheum palmatum L. including aloe-emodin, rhein, emodin, chrysophannol-8-O-glucophranoside and physionl-8-O-glucophranoside were active in binding to the immobilized DNA.
Subject(s)
Chromatography, Affinity/methods , DNA/chemistry , Drugs, Chinese Herbal/analysis , Chromatography, High Pressure Liquid/methods , Drugs, Chinese Herbal/isolation & purification , Mass Spectrometry/methodsABSTRACT
Traditional Chinese medicines (TCMs) are attracting increased global attention because of their potential to provide novel therapeutic agents based on substantial historical records of efficacy in man. Many strategies have been designed for the screening and selection of bioactive compounds from these complex natural products mixtures. Biological fingerprinting analysis (BFA), based on small molecule-biomacromolecule interactions in complex systems, has been applied to screen the multiple bioactive compounds in natural products. Here we review the chromatographic and MS approaches used for BFA of natural products with targeting absorption, distribution, metabolism, elimination and toxicity (ADME/Tox) properties. Such chromatographic methods cover a wide range of applications including liposome, serum proteins, liver homogenate and DNA profiling. MS methods for the characterization of molecular interactions between natural products and target molecules by ESI and MALDI-TOF MS are also discussed.
Subject(s)
Chromatography, Liquid/methods , Medicine, Chinese Traditional , Spectrometry, Mass, Electrospray Ionization/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Biological Products/chemistryABSTRACT
A mode of comprehensive 2-D LC was developed by coupling a silica-bonded HSA column to a silica monolithic ODS column. This system combined the affinity property of the HSA column and the high-speed separation ability of the monolithic ODS column. The affinity chromatography with HSA-immobilized stationary phase was applied to study the interaction of multiple components in traditional Chinese medicines (TCMs) with HSA according to their affinity to protein in the first dimension. Then the unresolved components retained on the HSA column were further separated on the silica monolithic ODS column in the second dimension. By hyphenating the 2-D separation system to diode array detector and MS detectors, the UV and molecular weight information of the separated compounds can also be obtained. The developed separation system was applied to analysis of the extract of Rheum palmatum L., a number of low-abundant components can be separated on a single peak from the HSA column after normalization of peak heights. Six compounds were preliminarily identified according to their UV and MS spectra. It showed that this system was very useful for biological fingerprinting analysis of the components in TCMs and natural products.
Subject(s)
Chromatography, High Pressure Liquid/methods , Rheum/chemistry , Chromatography, Affinity/methods , Drugs, Chinese Herbal/chemistry , Humans , Mass Spectrometry , Molecular Structure , Serum Albumin/chemistry , Silicon Dioxide , Spectrophotometry, UltravioletABSTRACT
A hyphenated method for the isolation and identification of components in a traditional Chinese medicine of Honeysuckle was developed. Ion-exchange chromatography (IEC) was chosen for the fractionation of Honeysuckle extract, and then followed by concentration of all the fractions with rotary vacuum evaporator. Each of the enriched fractions was then further analyzed by reversed-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometer (RPLC-APCI/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) with matrix of oxidized carbon nanotubes, respectively. It can be noted totally more than 117 components were detected by UV detector, APCI/MS and MALDI-TOF/MS in Honeysuckle extract except the 145 components identified by MALDI-TOF/MS alone with this integrated approach, and 7 of them were preliminary identified according to their UV spectra and mass spectra performed by APCI/MS and MALDI-TOF/MS, respectively. The obtained analytical results not only indicated the approach of integration IEC fractionation with RPLC-APCI/MS and MALDI-TOF/MS is capable of analyzing complex samples, but also exhibited the potential power of the mass spectrometer in detection of low-mass compounds, such as traditional Chinese medicines (TCMs) and complex biological samples.
Subject(s)
Lonicera/chemistry , Chromatography, Ion Exchange , Chromatography, Liquid , Indicators and Reagents , Mass Spectrometry , Plant Extracts/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spectrophotometry, UltravioletABSTRACT
A new strategy for screening of antineoplastic components in the traditional Chinese medicine of Radix et Rhizoma Rhei has been developed using chromatographic fingerprints before and after metabolism by high performance liquid chromatography-mass spectrometry. The metabolizing method was established based on the in vitro metabolism by Sprague-Dawley (SD) rat liver homogenate. By means of the metabolism methods in vitro, the antineoplastic activity of the extracts, metabolites and components of Radix et Rhizoma Rhei were determined by microculture tetrazolium (MTT) assays in vitro. It was observed that the inhibition rate of the crude extract for HeLa cell was increased from 26.7% to 36.2% after 60 min of metabolism. The changes of activities resulted from the changes of components' structures and the bioactive components were discovered simultaneously in view of metabolism by inhibiting rate assay for the components in Radix et Rhizoma Rhei. It is concluded that the antineoplastic activity of the crude extract from Radix et Rhizoma Rhei was increased after in vitro metabolism because the antineoplastic activity of aloe-emodin, the metabolite of chrysophanol, is higher than its parent compounds.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/pharmacokinetics , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/pharmacokinetics , Rheum/chemistry , Animals , Anthraquinones/chemistry , Anthraquinones/pharmacokinetics , Anthraquinones/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Cell Survival/drug effects , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal/chemistry , HeLa Cells , Humans , Male , Mass Spectrometry , Rats , Rats, Sprague-DawleyABSTRACT
Performance of comprehensive two-dimensional liquid chromatography system is greatly improved than we reported previously by using a silica monolithic column as for the second dimensional separation. Due to the increase of the elution speed on the second dimensional monolithic column, the first dimensional column efficiency and analysis rate can be greatly improved as comparing with conventionally second dimensional column. The developed system was applied to analysis of methanol extraction of two umbelliferae herbs Ligusticum chuanxiong Hort. and Angelica sinensis (Oliv.) Diels by using CN column as for the first dimensional separation and a silica monolithic ODS column for the second dimensional separation, and the obtained three-dimensional chromatograms were treated by normalization of peak heights with the value of the highest peak or setting a certain value using a software written in-house. It was observed that much more peaks for low-abundant components in TCM extract can clearly be detected here than we reported before, due to the large difference for the amount of components in TCMs' extract. With the above improvements in separation performance and data treatment, totally about 120 components in methanol extraction of Rhizoma chuanxiong and 100 in A. sinensis were separated with UV detection within 130 min. This result meant that both the number of peaks detected increase twice but the analysis time decrease twice if comparing with the previously reported result.