Determination of dopamine and its relativity of baicalin in rat nuclei after intravenous administration of flavonoids from Scutellariae radix

Baicalin is an active component of Scutellariae radix extracts. It can cross the blood-brain barrier and distribute in cerebral nuclei. However, its mechanism and the effects on the nuclei where it accumulates remain unclear. We used an HPLC-electrochemical detection method to determine the dopamine in cerebral nuclei after intravenous administration of flavonoids from Scutellariae radix and analyzed the relativity between baicalin and DA in cerebral nuclei. We found that the dopamine system is another target system of brain function which baicalin acts on.     

Analysis of baicalein, baicalin and wogonin in Scutellariae radix and its preparation by microemulsion electrokinetic chromatography with 1-butyl-3-methylimizolium tetrafluoborate ionic liquid as additive

Microemulsion electrokinetic chromatography (MEEKC) using 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) ionic liquid (IL) as additive was developed for the analysis of baicalin, wogonin and baicalein in Scutellariae radix and its preparation. After conducting a series of optimizations, baseline separation was obtained for the analytes within 5min under the optimum conditions (sodium dodecyl sulfate (SDS) 0.88% (m/v) ethyl acetate 0.8% (v/v) butan-1-ol 0.2% (v/v) and the buffer composition were 25% acetonitrile (v/v), 7.5 mM BMIM-BF4 and 10 mM NaH2PO4, pH 8.2, applied voltage 17.5 kV and detection at 254 nm), the method has been successfully applied to the determination and quantification of the analytes in the extracts of S. radix (cooked), S. radix (raw) and Qingfeiyihuowan which was the preparation including S. radix.     

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome and peptide profiling analysis by using a monolithic analytical capillary column

An automated nano-LC-MS/MS platform without trap column was established, which only used a 20 cm lauryl methacrylate-ethylene dimethacrylate (LMA-EDMA) monolithic capillary column to allow preconcentration and separation of peptides. The monolithic column had the advantages of good permeability and low backpressure resulting in higher flow rates for capillary columns. Tryptic digests of bovine albumin and yeast protein extract were tested using the monolithic column system. High proteomic coverage using this approach were demonstrated in this study. Furthermore, peptide samples extracted from mouse liver were separated by using the monolithic column system combined with size-exclusion chromatography prefractionation. This monolithic column system might be a promising alternative for the automated system previously using a trap column for routine proteome and peptide profiling analysis.     

A label-free differential proteomic analysis of mouse bronchoalveolar lavage fluid exposed to ultrafine carbon black

Ultrafine carbon black (ufCB) is a potential hazard to the lung. It causes changes in protein expression and it increases alveolar-capillary permeability in the lung. Label-free quantitative proteomic methods allow a sensitive and accurate analytical method for identifying and quantifying proteins in a protein mixture without chemically modifying the proteins. We used a label-free quantitative proteomic approach that combined and aligned LC-MS and LC-MS/MS spectra to analyze mouse bronchoalveolar lavage fluid (BALF) protein changes associated with exposure to ufCB. We developed a simple normalization method for quantification without spiking the internal standard. The intensities of unchanged peptides were used as normalization factors based on a statistical method to avoid the influence of peptides changed because of ufCB. LC-MS/MS spectra and then database searching were used to identify proteins. The relative abundances of the aligned peptides of identified proteins were determined using LC-MS spectra. We identified 132 proteins, of which 77 are reported for the first time. In addition, the expression of 15 inflammatory proteins and surfactant-associated proteins was regulated (i.e., 7 upregulated and 8 downregulated) compared with the controls. Several proteins not previously reported provide complementary information on the proteins present in mouse BALF, and they are potential biomarkers for the understanding of mechanisms involved in ufCB-induced lung disorders hypothesize that using the label-free quantitative proteomic approach introduced here is well suited for more rigorous, large-scale quantitative analysis of biological samples. We hypothesize that this label-free quantitative proteomic approach will be suited for a large-scale quantitative analysis of biological samples.     

Prerequisites for peptidomic analysis of blood samples: II. Analysis of human plasma after oral glucose challenge -- a proof of concept

Mass spectrometric plasma analysis for biomarker discovery has become an exploratory focus in proteomic research: the challenges of analyzing plasma samples by mass spectrometry have become apparent not only since the human proteome organization (HUPO) has put much emphasis on the human plasma proteome. This work demonstrates fundamental proteomic research to reveal sensitivity and quantification capabilities of our Peptidomics technologies by detecting distinct changes in plasma peptide composition in samples after challenging healthy volunteers with orally administered glucose. Differential Peptide Display (DPD) is a technique for peptidomics studies to compare peptides from distinct biological samples. Mass spectrometry (MS) is used as a qualitative and quantitative analysis tool without previous trypsin digestion or labeling of the samples. Circulating peptides (< 15 kDa) were extracted from 1.3 mL plasma samples and the extracts separated by liquid chromatography into 96 fractions. Each fraction was subjected to MALDI MS, and mass spectra of all fractions were combined resulting in a 2D-display of > 2,000 peptides from each sample. Endogenous peptides that responded to oral glucose challenge were detected by DPD of pre-and post-challenge plasma samples from 16 healthy volunteers and subsequently identified by nESI-qTOF MS. Two of the 15 MS peaks that were significantly modulated by glucose challenge were subsequently identified as insulin and C-peptide. These results were validated by using immunoassays for insulin and C-peptide. This paper serves as a proof of principle for proteomic biomarker discovery down to the pM concentration range by using small amounts of human plasma.     

Qualitative and quantitative analysis of toosendanin in Melia toosendan Sieb. Et Zucc (Meliaceae) with liquid chromatography/tandem mass spectrometry

Toosendanin (TSN) is a triterpenoid derivative found in Melia toosendan Sieb. Et Zucc (Meliaceae) or chinaberry. TSN present in the medicinal plants was first isolated and established by spectroscopic methods. In this report, high-performance liquid chromatography (HPLC) separation using columns of smaller particle size with tandem mass spectrometry (MS(n)) was used for the rapid determination of TSN in botanical extracts. A comparison of different fragmentation patterns shows that the results from positive and negative ion electrospray ionization (ESI)-MS(n) are complementary. The two modes can yield structurally significant information for the characterization and rapid identification of TSN in botanical extracts. The data obtained showed that MS(3) generated more characteristic ions that are useful for the identification of TSN in unknown samples. The separation of TSN was achieved with a water/acetonitrile gradient system using a short C18 reversed-phase column with small particle size (50 x 2.0 mm, 3.5 microm). With LC/MS, the quantitative analysis of TSN in the botanical extracts was done using external standard calibration and the method precision was found to vary from 4.3 to 7.6% (RSD, n = 5) on different days.     

Proteomic Analysis of the Protein Expression Profile in the Mature <Emphasis Type="Italic">Nigella sativa</Emphasis> (Black Seed)

Nigella sativa (N. sativa) seed has been used as an important nutritional flavoring agent and in traditional medicine for treating many illnesses since ancient times. Understanding the proteomic component of the seed may lead to enhance the understanding of its structural and biological functional complexity. In this study, we have analyzed its proteome profile based on gel-based proteome mapping technique that includes one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry strategy. We have not come across any such studies that have been performed in N. sativa seeds up to date. A total of 277 proteins were identified, and their functional, metabolic, and location-wise annotations were carried out using the UniProt database. The majority of proteins identified in the proteome dataset based on their function were those involved in enzyme catalytic activity, nucleotide binding, and protein binding while the major cellular processes included regulation of biological process followed by regulation of secondary biological process, cell organization and biogenesis, protein metabolism, and transport. The identified proteome was localized mainly to the nucleus then to the cytoplasm, plasma membrane, mitochondria, plastid, and others. A majority of the proteins were involved in biochemical pathways involving carbohydrate metabolism, amino acid and shikimate pathway, lipid metabolism, nucleotide, cell organization and biogenesis, transport, and defense processes. The identified proteins in the dataset help to improve our understanding of the pathways involved in N. sativa seed metabolism and its biochemical features and detail out useful information that may help to utilize these proteins. This study could thus pave a way for future further high-throughput studies using a more targeted proteomic approach.     

凝胶渗透色谱净化超高效液相色谱-串联质谱法检测甘草及其提取物中的11种氨基甲酸酯类农药残留

建立了甘草及其提取物中11种氨基甲酸酯类农药多残留的超高效液相色谱-串联质谱(UPLC-MS/MS)分析方法。以11种氨基甲酸酯类农药为目标分析物,样品经乙腈超声提取、凝胶渗透色谱(GPC)净化后,用UPLC-MS/MS检测。以甘草及其提取物为例,分别进行了0.02、0.04和0.1 mg/kg 3个添加浓度的11种目标分析物的加标回收率实验,甘草中11种目标分析物的回收率为72.2%～94.0%,相对标准偏差为0.7%～7.8%;甘草提取物中11种目标分析物的回收率为73.8%～94.7%,相对标准偏差为1.5%～12.7%。该方法灵敏度高、准确度好,符合农药多残留检测的技术要求,适用于甘草、黄芪等中药材及其提取物中氨基甲酸酯类农药残留的检测。     

The impact of blood on liver metabolite profiling – a combined metabolomic and proteomic approach

Metabolomics has entered the well‐established omic sciences as it is an indispensable information resource to achieve a global picture of biological systems. The aim of the present study was to estimate the influence of blood removal from mice liver as part of sample preparation for metabolomic and proteomic studies. For this purpose, perfused mice liver tissue (i.e. with blood removed) and unperfused mice liver tissue (i.e. containing blood) were compared by two‐dimensional gas chromatography time of flight mass spectrometry (GC × GC‐TOFMS) for the metabolomic part, and by liquid chromatography tandem mass spectrometry (LC‐MS/MS) for the proteomic part. Our data showed significant differences between the unperfused and perfused liver tissue samples. Furthermore, we also observed an overlap of blood and tissue metabolite profiles in our data, suggesting that the perfusion of liver tissue prior to analysis is beneficial for an accurate metabolic profile of this organ. Copyright © 2013 John Wiley & Sons, Ltd.     

Pressurized hot water extraction of berberine, baicalein and glycyrrhizin in medicinal plants

Pressurized hot water extraction (PHWE) using a laboratory made system was applied for the extraction of thermally labile and reasonably polar components such as berberine in coptidis rhizoma, glycyrrhizin in radix glycyrrhizae/liquorice and baicalein in scutellariae radix. PHWE was carried out dynamically at a flow of 1 ml/min, temperature between 95 and 140 °C, an applied pressure of 10-20 bar and extraction time of 40 min. Extraction by PHWE was found to give efficiencies comparable to Soxhlet extraction for baicalein in scutellariae radix and sonication for berberine in coptidis rhizoma, and glycyrrhizin in radix glycyrrhizae. Effects of ethanol added into the water used in PHWE were explored. Pressurized liquid extraction (PLE) with methanol as solvent was used for extraction of baicalein in scutellariae radix. The marker compounds present in the various medicinal plant extracts were determined by gradient elution HPLC.     

Proteome analysis of human dorsolateral prefrontal cortex using shotgun mass spectrometry

The prefrontal cortex executes important functions such as differentiation of conflicting thoughts, correct social behavior and personality expression, and is directly implicated in different neurodegenerative diseases. We performed a shotgun proteome analysis that included IEF fractionation, RP-LC, and MALDI-TOF/TOF mass spectrometric analysis of tryptic digests from a pool of seven human dorsolateral prefrontal cortex protein extracts. In this report, we present a catalog of 387 proteins expressed in these samples, identified by two or more peptides and high confidence search scores. These proteins are involved in different biological processes such as cell growth and/or maintenance, metabolism/energy pathways, cell communication/signal transduction, protein metabolism, transport, regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism, and immune response. This analysis contributes to the knowledge of the human brain proteome by adding sample diversity and protein expression data from an alternative technical approach. It will also aid comparative studies of different brain areas and medical conditions, with future applications in basic and clinical research.     

Shotgun proteomic analysis of microdissected postmortem human pituitary using complementary two-dimensional liquid chromatography coupled with tandem mass spectrometer

The pituitary is responsible for multiple homeostatic functions including metabolism, growth and reproduction. Proteome analysis offers an efficient approach for a comprehensive analysis of pituitary protein expression. The pituitary is usually acquired from postmortem specimens, which may potentially affect the proteome profile by proteolysis. The aim of this study was to determine whether the postmortem pituitary could be used in proteomic analysis combining with Laser capture microdissection (LCM). Digested peptides from LCM captured prolactin (PRL) cells were separated by two dimensional-nanoscale liquid chromatography (2D-nanoLC/MS) and characterized by tandem mass spectrometry (MS). All MS/MS spectrums were searched by SEQUEST and a proteome of 1660 proteins was identified. Category analysis of the proteome revealed an extensive unbiased access to cell component proteins with diverse functional characteristics. The results demonstrated the ability of using 2D-nanoLC/MS to perform sensitive proteomic analysis on limited protein quantities through microdissection. Detailed comparisons between the proteome in question and the one derived from the prolactinoma controls at peptide and protein levels indicated that the two proteomes had similar characters. Overall, our results revealed for the first time the possibility of use of postmortem human pituitary for proteomic research which is important for further studies on disease biomarker identification and molecular mechanisms of prolactinoma tumorigenesis.     

Relative quantitation of peptides in wild-type and Cpe(fat/fat) mouse pituitary using stable isotopic tags and mass spectrometry

Cpe(fat/fat) mice have a point mutation in the coding region of the carboxypeptidase E gene that renders the enzyme inactive. As a result, these mice have reduced levels of several neuropeptides and greatly increased levels of the peptide processing intermediates that contain C-terminal basic residues. However, previous studies examined a relatively small number of neuropeptides. In the present study, we used a quantitative peptidomics approach with stable isotopic labels to examine the levels of pituitary peptides in Cpe(fat/fat) mice relative to wild-type mice. Pituitary extracts from mutant and wild type mice were labeled with the stable isotopic label [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propyl]trimethylammonium chloride containing nine atoms of hydrogen or deuterium. Then, the two samples were pooled and analyzed by liquid chromatography/mass spectrometry (LC/MS). The relative abundance of peptides was determined from a comparison of the intensities of the heavy and light peaks. Altogether, 72 peptides were detected in the Cpe(fat/fat) and/or wild-type mouse pituitary extracts of which 53 were identified by MS/MS sequencing. Several peptides identified in this analysis represent previously undescribed post-translational processing products of known pituitary prohormones. Of the 72 peptides detected in pituitary, 17 were detected only in the Cpe(fat/fat) mouse extracts; these represent peptide processing intermediates containing C-terminal basic residues. The peptides common to both Cpe(fat/fat) and wild-type mice were generally present at 2-5-fold lower levels in the Cpe(fat/fat) mouse pituitary extracts, although some peptides were present at equal levels and one peptide (acetyl beta-endorphin 1-31) was increased approximately 7-fold in the Cpe(fat/fat) pituitary extracts. In contrast, acetyl beta-endorphin 1-26 was present at approximately 10-fold lower levels in the Cpe(fat/fat) pituitary, compared with wild-type mice. The finding that many peptides are substantially decreased in Cpe(fat/fat) pituitary is consistent with the broad role for carboxypeptidase E in the biosynthesis of numerous neuropeptides.     

高通量蛋白质组学分析研究进展

基于质谱的蛋白质组学技术已经日趋成熟,可以对细胞和组织中的成千上万种蛋白质进行全面的定性和定量分析,逐步实现“深度覆盖”。随着生物医学日益增长的大队列蛋白质组学分析需求,如何在保持较为理想的覆盖深度下实现短时间、快速的“高通量”蛋白质组学分析已成为当前亟需解决的关键问题之一。常规的蛋白质组学分析流程通常包括样品前处理、色谱分离、质谱检测和数据分析。该文从以上4个方面展开介绍近10年以来高通量蛋白质组学分析技术取得的一系列研究进展,主要包括:(1)基于高通量、自动化移液工作站的蛋白质组样品前处理方法;(2)基于微升流速液相色谱与质谱联用的高通量蛋白质组检测方法;(3)利用灵敏度高、扫描速度快的质谱仪实现短色谱梯度分离下蛋白质组深度覆盖的分析方法;(4)基于人工智能、深度神经网络、机器学习等的蛋白质组学大数据分析方法。此外,对高通量蛋白质组学面临的挑战及其发展进行展望。总而言之,预期在不久的将来高通量蛋白质组学技术将会逐步“落地转化”,成为大队列蛋白质组学分析的利器。     

Imaging mass spectrometry with silver naeoparticles reveals the distribution of fatty acids in mouse retinal sections

A new approach to the visualization of fatty acids in mouse liver and retinal samples has been developed using silver nanoparticles (AgNPs) in nanoparticle-assisted laser desorption/ ionization imaging mass spectrometry (nano-PALDI-IMS) in negative ion mode. So far, IMS analysis has concentrated on main cell components, such as cell membrane phospholipids and cytoskeletal peptides. AgNPs modified with alkylcarboxylate and alkylamine were used for nano-PALDI-IMS to identify fatty acids, such as stearic, oleic, linoleic, arachidonic, and eicosapentaenoic acids, as well as palmitic acid, in mouse liver sections; these fatty acids are not detected using 2,5-dihydroxybenzoic acid (DHB) as a matrix. The limit of detection for the determination of palmitic acid was 50 pmol using nano-PALDI-IMS. The nano-PALDI-IMS method is successfully applied to the reconstruction of the ion images of fatty acids in mouse liver sections. We verified the detection of fatty acids in liver tissue sections of mice by analyzing standard lipid samples, which showed that fatty acids were from free fatty acids and dissociated fatty acids from lipids when irradiated with a laser. Additionally, we applied the proposed method to the identification of fatty acids in mouse retinal tissue sections, which enabled us to learn the six-zonal distribution of fatty acids in different layers of the retina. We believe that the current approach using AgNPs in nano-PALDI-IMS could lead to a new strategy to analyze basic biological mechanisms and several diseases through the distribution of fatty acids.     

Proteomics based on high-efficiency capillary separations

Identifying and quantifying in a high throughput manner the proteins expressed by cells, tissues or an organism provides the basis for understanding the functions of its constituents at a "systems" level. As a result, proteome analysis has increasingly become the focus of significant interest and research over the past decade. This is especially true following the recent stunning achievements in genomics analyses. However, unlike the static genome, the complexities and dynamism of the proteome present significant analytical challenges and demand highly efficient separations and detection technologies. A number of recent technological advancements have been in direct response to these challenges. Currently, strategically mated combinations of sophisticated separations techniques and advanced mass spectrometric detection represent the best approach to addressing the intricacies of the proteome. Liquid-phase separations, often within capillaries, are increasingly recognized as the best separations technique for this approach. In combination on-line with mass spectrometry, liquid-phase separations provide the improved analytical sensitivity, sample throughput, and quantitation capabilities necessitated by the multifaceted problems within proteomics analyses. This review focuses primarily on current high-efficiency capillary separations techniques, including both capillary liquid chromatography and capillary electrophoresis, applied to the analysis of complex proteomic samples. We emphasize developments at our laboratory and illustrate technical advances that attempt to review the role of separations within the broader context of a state-of-the-art integrated proteomics effort.     

基于UPLC/Q-TOF-MS分析附子半夏配伍相反的物质基础

利用超高效液相色谱-飞行时间质谱联用技术(UPLC/Q-TOF-MS)分析附子半夏药对配伍相反的物质基础,从化学成分层次阐释其配伍相反机制.基于UPLC/Q-TOF-MS建立附子半夏药对配伍后生物碱类成分的化学指纹图谱,通过主成分分析法和正交偏最小二乘判别法分析药对配伍在合煎过程中的生物碱类成分的含量变化,找出差异变化显著的化学成分.结果表明正离子模式时附子半夏药对合煎液中次乌头碱,中乌头碱,乌头碱,去氧乌头碱,10-OH-中乌头碱,10-OH-乌头碱等的含量明显增高,而中乌头原碱,去乙酸中乌头原碱,去乙酸次乌头原碱,苯甲酰乌头原碱,苯甲酰次乌头原碱,10-OH-苯甲酰中乌头原碱等含量降低.附子半夏药对配伍应用时双酯型二萜生物碱的含量明显增高,而单酯型二萜生物碱的含量明显降低,这可能是附子半夏药对配伍相反作用的物质基础.     

Efficient discovery and capture of new neuronal nitric oxide synthase–postsynaptic density protein‐95 uncouplers from herbal medicines using magnetic molecularly imprinted polymers as artificial antibodies

In the scope of stroke treatment, new neuronal nitric oxide synthase–postsynaptic density protein‐95 uncouplers from herbal medicines were discovered and captured. To do so, highly selective magnetic molecularly imprinted polymers with a core–shell structure were prepared as artificial antibodies. According to the results of computational simulations, we designed and synthesized various polymers with varying amounts and types of template, functional monomer, cross‐linker, and solvent. Characterization and performance tests revealed that the most appropriate artificial antibodies showed uniform spherical morphologies, large adsorption capacities, fast‐binding kinetics, high selectivity, and quick separation. These artificial antibodies were then used as sorbents for dispersive magnetic solid‐phase extraction coupled with high‐performance liquid chromatography and mass spectrometry to capture and identify structural analogs to ZL006 from extracts of Scutellariae radix, Psoraleae fructus, and Trifolium pratense. Furthermore, according to the neuroprotective effect and coimmunoprecipitation test, Baicalein, Neobavaisoflavone, Corylifol A, and Biochanin A can be the potential uncouplers of neuronal nitric oxide synthase–postsynaptic density protein‐95. Therefore, this present study contributes valuable information for the discovery of neuronal nitric oxide synthase–postsynaptic density protein‐95 uncouplers from herbal medicines.     

Mapping the human plasma proteome by SCX-LC-IMS-MS

The advent of on-line multidimensional liquid chromatography-mass spectrometry has significantly impacted proteomic analyses of complex biological fluids such as plasma. However, there is general agreement that additional advances to enhance the peak capacity of such platforms are required to enhance the accuracy and coverage of proteome maps of such fluids. Here, we describe the combination of strong-cation-exchange and reversed-phase liquid chromatographies with ion mobility and mass spectrometry as a means of characterizing the complex mixture of proteins associated with the human plasma proteome. The increase in separation capacity associated with inclusion of the ion mobility separation leads to generation of one of the most extensive proteome maps to date. The map is generated by analyzing plasma samples of five healthy humans; we report a preliminary identification of 9087 proteins from 37,842 unique peptide assignments. An analysis of expected false-positive rates leads to a high-confidence identification of 2928 proteins. The results are catalogued in a fashion that includes positions and intensities of assigned features observed in the datasets as well as pertinent identification information such as protein accession number, mass, and homology score/confidence indicators. Comparisons of the assigned features reported here with other datasets shows substantial agreement with respect to the first several hundred entries; there is far less agreement associated with detection of lower abundance components.