摘要：
Although much effort has been made in the field of membrane proteomics, the analysis of membrane proteins particularly integral membrane proteins with poor water solubility still presents a great challenge. In this paper, 2% SDS was used to extract membrane proteins and experimental conditions for the application of acetone precipitation method to the cleanup of SDS-solubilized membrane protein sample were optimized. For improving the re-dissolution and trypsinolysis of acetone-precipitated proteins, several commonly used additives, urea, methanol and sodium deoxycholate (SDC), were employed and compared. The results showed that, when the pre-cooled acetone-to-sample ratio was 6:1 (v/v) with one additional washing step, residual SDS in the protein sample could be lowered to below 0.01% and more than 90% of the proteins were precipitated and therefore recovered. 1% SDC-containing buffer could improve the re-dissolution and digestion of the acetone precipitated proteins more efficiently than the others. Using the combinative sample preparation strategy developed, 398 proteins were identified from the rat liver membrane-enriched fraction, including 188 membrane proteins. Compared with other three representative solution-based sample preparation methods commonly used in membrane proteomics, the newly developed combinative strategy increased the number of identified total proteins and membrane proteins on average by 29.2% and 28.5%, respectively. This combinative strategy was demonstrated to be easily operated at low cost and suitable for the analysis of membrane proteins varying in type and sample volume, etc. (C) 2012 Published by Elsevier B.V.
摘要：
BACKGROUND: Shotgun proteomics data analysis usually relies on database search. Because commonly employed protein sequence databases of most species do not contain sufficient protein information, the application of shotgun proteomics to the research of protein sequence profile remains a big challenge, especially to the species whose genome has not been sequenced yet. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we present a workflow with integrated database to partly address this problem. First, we downloaded the homologous species database. Next, we identified the transcriptome of the sample, created a protein sequence database based on the transcriptome data, and integtrated it with homologous species database. Lastly, we developed a workflow for identifying peptides simultaneously from shotgun proteomics data. CONCLUSIONS/SIGNIFICANCE: We used datasets from orange leaves samples to demonstrate our workflow. The results showed that the integrated database had great advantage on orange shotgun proteomics data analysis compared to the homologous species database, an 18.5% increase in number of proteins identification.
作者机构：
[Wang, Xianchun; Yan, Yizhong; Chen, Ping; He, Quanze; Liu, Yi; Li, Jianjun; Lin, Yong; Liang, Songping] Hunan Normal Univ, Coll Life Sci, Key Lab Prot Chem & Dev Biol, Natl Educ Comm, Changsha 410081, Hunan, Peoples R China.;[Lin, Yong] Hunan Agr Univ, Natl Res Ctr Engn & Technol Utilizat Bot Funct In, Coll Hort & Landscape, Changsha, Hunan, Peoples R China.
通讯机构：
[Wang, Xianchun] H;Hunan Normal Univ, Coll Life Sci, Key Lab Prot Chem & Dev Biol, Natl Educ Comm, Changsha 410081, Hunan, Peoples R China.
关键词：
Membrane proteome;Protein fractionation;Sample cleanup;SDS;Special gradient gel
摘要：
SDS is mostly used to enhance the solubilization and extraction of membrane proteins due to its strong detergency and low cost. Nevertheless, SDS interferes with the subsequent procedures and needs to be removed from the samples. In this work, a special gradient gel electrophoresis (GGE) system was developed to remove SDS from the SDS-solubilized protein samples. As a proof-of-principle experiment, the GGE system was designed to be composed of an agarose loading layer, six polyacrylamide fractionation layers with different concentrations and a high-concentration polyacrylamide sealing layer. The advantages of the GGE system are that it not only can electrophoretically remove SDS efficiently so that the protein loss resulted from the repeated gel washing after electrophoresis was avoided, but also can reduce the complexity of the sample, prevent the precipitation of proteins after loading and avoid the loss of proteins with low molecular weight during the electrophoresis. Using GGE system, about 85% of SDS in the sample and gel was electrophoretically removed and the proteins were fractionated. Compared with the two representative gel-based sample cleanup methods reported in literature, GGE-based strategy significantly improved the identification efficiency of proteins in terms of the number and coverage of the identified proteins.
摘要：
The study of DNA-binding proteins is crucial in understanding gene regulatory networks. We developed a new method for the enrichment of DNA-binding proteins based on the variability of DNA-protein complexes’ solubility in different ionic strength solutions. 0.14M sodium chloride was determined as the most efficient extraction concentration to precipitate DNA-binding proteins. SDS-PAGE analysis revealed that some high-abundance proteins were removed effectively and at the same time DNA-binding proteins were isolated in this simple process. Twenty kinds of proteins were identified in the acquired sample by 1-D gel-LC-MS/MS. Furthermore, computerized analysis of MS data showed that quite a number of unmatched peptides have the classic structure of leucine zipper or zinc finger, which were symbolic elements of transcription factors. These results suggested that this new method can acquire DNA-binding proteins effectively and allow improvement in the isolation of high-quality DNA-binding proteins.
摘要：
It is still a major challenge to simultaneously isolate artemisinin and its precursors, especially dihydroartemisinic acid and artemisinic acid, from herbal Artemisia annua. A rapid, economical and automatical chromatographic separation process to isolate and purify artemisinin, dihydroartemisinic acid and artemisinic acid at the same time on a preparative scale was developed. The procedure included solvent extraction of ground Artemisia annua leaves by refluxing and purification of crude extract by preparative reverse-phase high-performance liquid chromatography (RP-HPLC). Fractions containing artemisinin and its precursors were collected and identified by gas chromatography and mass spectrometry. High purity of artemisinin, dihydroartemisinic acid and artemisinic acid was obtained by preparative HPLC with a C18 column and 60% acetonitrile in water as the mobile phase. The techniques described here are useful tools for the preparative-scale isolation of artemisinin and its precursors in a fast, cost-effective and environmental friendly manner. Copyright (C) 2011 John Wiley & Sons, Ltd.
摘要：
Two new cinnamic acids, 2-O-caffeoyl-3-O-isoferuloyltartaric (3), and 2, 3-di-O-isoferuloyltartaric acid (5), along with three known caffeic acids, cichoric acid (1), 2-O-caffeoyl-3-O-feruloyltartaric acid (2) and 2-O-caffeoyl-3-O-p-coumaroyltartaric acid (4), have been successfully isolated and purified from Echinacea purpurea. In this study, we investigated an efficient method for the preparative isolation and purification of cinnamic acids from E. purpurea by high-speed counter-current chromatography (HSCCC). The separation was performed using a two-phase solvent composed of n-hexane-ethyl-acetatemethanol- 0.5% aqueous acetic acid (1:3:1:4, v/v). The upper phase was used as the stationary phase and the lower phase as the mobile phase, with a flow rate of 1.6 mL/min. From 250 mg of crude extracts, 65.1 mg of 1, 8.3 mg of 2, 4.0 mg of 3, 4.5 mg of 4, and 4.3 mg of 5 were isolated in one-step, with purities of 98.5%, 97.7%, 94.6%, 94.3%, and 98.6%, respectively, as evaluated by HPLC-DAD. The chemical structures were identified by electro spray ionization mass spectrometry (ESI-MS) and one- and two-dimensional NMR spectra. HSCCC was very efficient for the separation and purification of the cinnamic acids from E. purpurea.
摘要：
Analysis of the membrane proteins, particularly the integral membrane proteins, is limited by the inherent membrane hydrophobicity. Sodium dodecyl sulfate (SDS) is one of the most efficient reagents used for the extraction of membrane proteins, but its presence in samples interferes with LC-MS-based proteomic analyses because it affects RP-LC separations and electrospray ionization. In this paper, we present an improved sample preparation strategy based on SOS-assisted digestion and peptide-level SDS-removal using an optimized potassium dodecyl sulfate (KDS) precipitation method (SSDP method) for shotgun analysis of the membrane proteome. This method utilizes a high concentration of SDS (1.0%) to lyse the membranes and to solubilize the hydrophobic membrane proteins, resulting in a more complete protein digestion in the diluted SDS buffer (0.1% SOS), and a high efficiency of SOS removal and peptide recovery by the optimized KDS precipitation for protein identification. The SSDP method provides evidence that proteins can be efficiently digested, and the SDS can be decreased to <0.01% allowing >95% peptide recovery. Compared to other sample preparation methods commonly used in shotgun membrane proteomics, the newly developed method not only increased the identified number of the total proteins, membrane proteins and integral membrane proteins by an average of 33.1%, 37.2% and 40.5%, respectively, but also leading to the identification of highest number of matching peptides. All the results showed that the method yielded better recovery and reliability in the identification of the proteins especially the highly hydrophobic integral membrane proteins, and thus providing a promising tool for the shotgun analysis of membrane proteome. (C) 2012 Elsevier B.V. All rights reserved.
摘要：
A non-aqueous solid phase extraction (SPE) method utilizing silica based strong cation exchange (SCX) was developed and optimized for the enrichment of alkaloids. In this method, silica based SCX SPE columns were used for the elimination of non-alkaloid compounds and the preconcentration of alkaloids from the extracts. Mass spectrometry was employed to analyze the alkaloid-enriched fraction, and results showed that the SPE method developed in this study was effective for the removal of non-alkaloids. Then, this pretreatment method was combined with high performance liquid chromatography for the quantification of scopolamine and hyoscyamine from Scopolia tangutica Maxim. The recoveries of scopolamine and (-)-hyoscyamine were 98.51% and 91.12%, respectively. Relative standard deviation values were 1.4% for scopolamine and 1.6% for (-)-hyoscyamine. The linearity was good in the 0.01-0.8 mg mL(-1) range for hyoscyamine and 0.01-0.4 mg mL(-1) range for scopolamine.
作者机构：
[龙文静] National Res. Center of Engineering Tech. for Utilization of Functional Ingredients from Botanicals, Changsha 410128, China;[袁玲] Key Laboratory of Tea Science of Ministry of Education, Hunan Agricultural University, Changsha 410128, China;Department of Botanical Resources Engineering, College of Horticulture and Gardening, Hunan Agricultural University, Changsha 410128, China;[李银花; 刘仲华] National Res. Center of Engineering Tech. for Utilization of Functional Ingredients from Botanicals, Changsha 410128, China, Key Laboratory of Tea Science of Ministry of Education, Hunan Agricultural University, Changsha 410128, China;[张盛] National Res. Center of Engineering Tech. for Utilization of Functional Ingredients from Botanicals, Changsha 410128, China, Department of Botanical Resources Engineering, College of Horticulture and Gardening, Hunan Agricultural University, Changsha 410128, China
通讯机构：
[Zhang, S.] N;National Res. Center of Engineering Tech. for Utilization of Functional Ingredients from Botanicals, Changsha 410128, China
关键词：
Anthraquinone;Debaryomyces hansenii;Denaturing gradient gel electrophoresis;Eurotium cristatum;Fungal community;Fuzhuan brick-tea
摘要：
Chinese Fuzhuan brick-tea is a unique microbial fermented tea characterized by a period of fungal growth during its manufacturing process. The aim of the present study was to characterize, both physicochemically and microbiologically, traditional industrial production processes of Fuzhuan brick-tea. Fermenting tea samples were collected from the largest manufacturer. Physicochemical analyses showed that the low water content in the tea substrates provided optimal growth conditions for xerophilic fungi. The fungal communities existing in tea materials, fermenting tea, and stored teas were monitored by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) targeting the D1 region of the 265 rRNA genes, followed by sequencing of the amplicons. Results revealed that the microorganisms were from, or closely related to, the genera Eurotium, Debaryomyces, Aspergillus, Verticillium, Pichia, Pestalotiopsis, Rhizomucor and Beauveria. This is the first report of Debaryomyces participating in the processing of Fuzhuan brick-tea. We concluded that the dominant genera Eurotium, Debaryomyces and Aspergillus are beneficial fungi associated with the fermentation of Fuzhuan brick-tea. The genus Beauveria was present in the stored Fuzhuan brick-tea, which may help protect tea products from insect spoilage. The remaining four genera were of minor importance in the manufacturing of Fuzhuan brick-tea. The predominant Eurotium species, a strain named Eurotium sp. FZ, was phenotypically and genotypically identified as Eurotium cristatum. High performance thin layer chromatography analysis of anthraquinones showed that emodin existed in all the dark tea samples, but physcion was only detectable in the tea fermented by E. cristatum. The PCR-DGGE approach was an effective and convenient means for profiling the fungal communities in Fuzhuan brick-tea. These results may help promote the use of microbial consortia as starter cultures to stabilize and improve the quality of Fuzhuan brick-tea products. (C) 2011 Elsevier B.V. All rights reserved.
摘要：
In-gel digestion is an attractive route in mass spectrometry-based proteomic analysis, which, however, often suffers from a certain amount of sample loss mainly due to insufficient protein digestion and peptide extraction. To address this, herein we establish a partially degradable gel-assisted protein digestion and peptide recovery method by means of a simple replacement of bis-acrylamide (BA) with bis-acrylylcystamine (BAC). Concretely, the protein sample solubilized using high concentrations of sodium dodecyl sulfate (SOS) and urea were directly entrapped and immobilized into BAC-crosslinked gel by vacuum-dried gel absorption followed by fixation treatment. After removal of SDS and urea by repeated washing, the proteins were subjected to in-gel digestion and the gel was reductively treated. The tryptic peptides were recovered from the partial degradation of the gel and analyzed afterwards by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). Compared with conventional BA-crosslinked gel method, this new method increased the numbers of identified proteins and unique peptides by 20.2% and 20.4%, respectively. The further statistical analysis demonstrated that the method improved the recovery of tryptic peptides particularly larger and/or hydrophobic peptides, thereby significantly facilitating protein identification. Thus, the newly developed method is a promising alternative for BA-crosslinked gel-based shotgun workflows and has potential application in the related fields of protein chemistry and proteomics. (C) 2011 Elsevier B.V. All rights reserved.
摘要：
Hexavalent chromium (Cr(VI)), a commonly used industrial metal, is a well-known mutagen and carcinogen, and occupational exposure can induce a broad spectrum of adverse health effects, including cancers. Although Cr(VI)-induced DNA damage is thought to be the primary mechanism of chromate genotoxicity and mutagenicity, there is an increasing number of reports showing that epigenetic mechanisms of gene regulation might be a central target of Cr(VI) toxicity. Epigenetic changes, such as changes in phosphorylation, altered DNA methylation status, histone acetylation and signaling pathways, have been observed after chromium exposure. Nevertheless, to better demonstrate the roles of epigenetic modifications in Cr(VI)-induced carcinogenesis, more work needs to be carried out. This study is aimed to investigate changes in biotinidase (BTD) and holocarboxylase synthetase (HCS), two major proteins which maintain homeostasis of the newfound epigenetic modification: histone biotinylation, in cells exposed to Cr(VI). The data showed that Cr(VI) decreased BID expression at the transcriptional level in human bronchial epithelial cells (16HBE). In addition, using the epigenetic modifiers, 5-Aza-2'-deoxycytidine (Aza) and Trichostatin A (TSA), we found that modifications of histone acetylation reversed the inhibition of BID, suggesting that Cr(VI) may cause down regulation of BID by modifications of histone acetylation. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
摘要：
A previous study has identified two types of recombinant variants of Potato virus Y strain NTN (PVYNTN) in China and sequenced the complete genome of the variant PVYNTN-HN2. In this study, the complete genome of isolate PVYNTN-HN1 was fully sequenced and analyzed. The most striking difference between the two variants was the location of recombinant joint three (RJ3). In PVYNTN-HN1, like other typical European-PVYNTN isolates such as PVYNTN-Hun, the RJ3 was located at nucleotide (nt) 9183, namely the 3' proximal end of the CP gene (nt. 8571-9371), thus leading to most (the first 613 nucleotides from the 5' proximal end) of the CP gene (801 bp) with a PVYN origin and PVYN-serotype; whereas in contrast, the RJ3 in PVYNTN-HN2 was located at nt 8572, consequently leading to a CP gene of PVYO origin and PVYO-serotype. The varied genome composition among PVYO, PVYN, PVYN:O, PVYNTN-HN1 and PVYNTN-HN2 made them useful for the investigation of possible roles of gene segment(s) in symptom formation on host plants. When Physalis floridana plants were infected with different PVY isolates, two types of symptoms were induced. PVYN and PVYNTN-HN1 induced mild symptoms (mainly mild mottling) whereas PVYO, PVYN:O and PVYNTN-HN2 induced serve symptoms including leaf and stem necrosis, leaf-drop and stunting. These results, together with a previous study using artificial PVY chimeras, demonstrate that the CP gene, especially the 5' proximal segment (nt 8572-9183), and/or CP likely determine the pathogenicity of PVY in P. floridana.
关键词：
Citrus canker disease;pthA-nls;Sense and antisense strands;Genetic transformation;Disease resistance;Sweet orange
摘要：
The COOH terminal of pthA encoding three nuclear localizing signals (NLS) was amplified by polymerase chain reaction (PCR) from the plasmid of Xanthomonas axonopodis pv. citri, the pathogen of citrus canker disease. Then the sense and antisense strands of the nls were cloned into pBI121 vector. pthA-nls driven by the CaMV35s promoter was transferred into sweet orange via Agrobacterium -mediated transformation. Successful integration was confirmed by PCR and Southern blotting, and 12 sense-nls (nls
+) and 9 antisense-nls (nls
−) transgenic clones were obtained. The expression of nls fragment was analyzed by RT–PCR, Real time q-PCR and Western blotting, in which the specific NLS protein was detected only in nls
+ transgenic clones. In an in vitro assay, when pin-puncture inoculation was performed with 2.5×107cfu/ml of bacterial solution, the nls
+ transgenic clones showed no typical lesion development, while typical symptoms were observed in the wild types and the nls
− transgenic clones. In vivo assay results indicated that the nls
+ transgenic clones showed less disease incidence, in comparison with the wild types and the nls
− transgenic clones, when pin-puncture inoculation was performed with 104–105cfu/ml. The minimum disease incidence was 23.3% for ‘Sucarri’ sweet orange and 33.3% for ‘Bingtang’ sweet orange. When 104–107cfu/ml of pathogen was spray inoculated, the nls
+ transgenic clones did not show any symptom, and even the concentration raised to 109cfu/ml, the disease incidence was 20–80%, while the wild types and the nls
− transgenic clones had 100% disease development with whatever concentration of inoculum. Two transgenic clones were confirmed to be resistant to citrus canker disease in the repeated inoculation. The results suggested that the transformation of nls sense strands may offer an effective way to acquire resistance to citrus canker disease.
