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1.
Non-specific lipid transfer proteins (nsLTPs) are common allergens and they are particularly widespread within the plant kingdom. They have a highly conserved three-dimensional structure that generate a strong cross-reactivity among the members of this family. In the last years several web tools for the prediction of allergenicity of new molecules based on their homology with known allergens have been released, and guidelines to assess potential allergenicity of proteins through bioinformatics have been established. Even if such tools are only partially reliable yet, they can provide important indications when other kinds of molecular characterization are lacking. The potential allergenicity of 28 amino acid sequences of LTPs homologs, either retrieved from the UniProt database or in silico deduced from the corresponding EST coding sequence, was predicted using 7 publicly available web tools. Moreover, their similarity degree to their closest known LTP allergens was calculated, in order to evaluate their potential cross-reactivity. Finally, all sequences were studied for their identity degree with the peach allergen Pru p 3, considering the regions involved in the formation of its known conformational IgE-binding epitope. Most of the analyzed sequences displayed a high probability to be allergenic according to all the software employed. The analyzed LTPs from bell pepper, cassava, mango, mungbean and soybean showed high homology (>70%) with some known allergenic LTPs, suggesting a potential risk of cross-reactivity for sensitized individuals. Other LTPs, like for example those from canola, cassava, mango, mungbean, papaya or persimmon, displayed a high degree of identity with Pru p 3 within the consensus sequence responsible for the formation, at three-dimensional level, of its major conformational epitope. Since recent studies highlighted how in patients mono-sensitized to peach LTP the levels of IgE seem directly proportional to the chance of developing cross-reactivity to LTPs from non-Rosaceae foods, and these chances increase the more similar the protein is to Pru p 3, these proteins should be taken into special account for future studies aimed at evaluating the risk of cross-allergenicity in highly sensitized individuals.  相似文献   

2.
Vitellogenin is a protein produced by the liver of oviparous animals in response to circulating estrogens. The amino acid sequence of vitellogenin from Atlantic cod (Gadus morhua) has not yet been determined. In this study Atlantic cod vitellogenin was characterized using a 'bottom-up' mass spectrometric approach. Vitellogenin synthesis was induced 'in vivo' with beta-estradiol, and subjected to trypsin digestion for characterization by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry. A peptide mass fingerprint was obtained and 'de novo' sequencing of the most abundant tryptic peptides was performed by low-energy collision-induced dissociation tandem mass spectrometry. As a result of these experiments, the sequences of various tryptic peptides have been elucidated. The database search has shown that Atlantic cod vitellogenin shares a series of common peptides with the two different known vitellogenin sequences of haddock, a closely related species. These findings allow us to propose that Atlantic cod might also co-express at least two distinct forms of vitellogenin.  相似文献   

3.
Limited proteolysis is an important and widely used method for analyzing the tertiary structure and determining the domain boundaries of proteins. Here we describe a novel method for determining the N- and C-terminal boundary amino acid sequences of products derived from limited proteolysis using semi-specific and/or non-specific enzymes, with mass spectrometry as the only analytical tool. The core of this method is founded on the recognition that cleavage of proteins with non-specific proteases is not random, but patterned. Based on this recognition, we have the ability to determine the sequence of each proteolytic fragment by extracting a common association between data sets containing multiple potential sequences derived from two or more different mass spectral molecular weight measurements. Proteolytic product sequences derived from specific and non-specific enzymes can be accurately determined without resorting to the conventional time-consuming and laborious methods of SDS-PAGE and N-terminal sequencing analysis. Because of the sensitivity of mass spectrometry, multiple transient proteolysis intermediates can also be identified and analyzed by this method, which allows the ability to monitor the progression of proteolysis and thereby gain insight into protein structures.  相似文献   

4.
In this work, partial characterization of the primary structure of phycocyanin from the cyanobacterium Aphanizomenon flos‐aquae (AFA) was achieved by mass spectrometry de novo sequencing with the aid of chemical derivatization. Combining N‐terminal sulfonation of tryptic peptides by 4‐sulfophenyl isothiocyanate (SPITC) and MALDI‐TOF/TOF analyses, facilitated the acquisition of sequence information for AFA phycocyanin subunits. In fact, SPITC‐derivatized peptides underwent facile fragmentation, predominantly resulting in y‐series ions in the MS/MS spectra and often exhibiting uninterrupted sequences of 20 or more amino acid residues. This strategy allowed us to carry out peptide fragment fingerprinting and de novo sequencing of several peptides belonging to both α‐ and β‐phycocyanin polypeptides, obtaining a sequence coverage of 67% and 75%, respectively. The presence of different isoforms of phycocyanin subunits was also revealed; subsequently Intact Mass Measurements (IMMs) by both MALDI‐ and ESI‐MS supported the detection of these protein isoforms. Finally, we discuss the evolutionary importance of phycocyanin isoforms in cyanobacteria, suggesting the possible use of the phycocyanin operon for a correct taxonomic identity of this species. Copyright © 2008 John Wiley & Sons, Ltd.  相似文献   

5.
A new strategy is described for the determination of amino acid sequences of unknown peptides. Different from the well-known but often inefficient de novo sequencing approach, the new method is based on a two-step process. In the first step the amino acid composition of an unknown peptide is determined on the basis of accurate mass values of the peptide precursor ion and a small number of accurate fragment ion mass values, and, as in de novo sequencing, without employing protein database information or other pre-information. In the second step the sequence of the found amino acids of the peptide is determined by scoring the agreement between expected and observed fragment ion signals of the permuted sequences. It was found that the new approach is highly efficient if accurate mass values are available and that it easily outstrips common approaches of de novo sequencing being based on lower accuracies and detailed knowledge of fragmentation behavior. Simple permutation and calculation of all possible amino acid sequences, however, is only efficient if the composition is known or if possible compositions are at least reduced to a small list. The latter requires the highest possible instrumental mass accuracy, which is currently provided only by fourier transform ion cyclotron resonance mass spectrometry. The connection between mass accuracy and peptide composition variability is described and an example of peptide compositioning and composition-based sequencing is presented.  相似文献   

6.
A number of different approaches have been described to identify proteins from tandem mass spectrometry (MS/MS) data. The most common approaches rely on the available databases to match experimental MS/MS data. These methods suffer from several drawbacks and cannot be used for the identification of proteins from unknown genomes. In this communication, we describe a new de novo sequencing software package, PEAKS, to extract amino acid sequence information without the use of databases. PEAKS uses a new model and a new algorithm to efficiently compute the best peptide sequences whose fragment ions can best interpret the peaks in the MS/MS spectrum. The output of the software gives amino acid sequences with confidence scores for the entire sequences, as well as an additional novel positional scoring scheme for portions of the sequences. The performance of PEAKS is compared with Lutefisk, a well-known de novo sequencing software, using quadrupole-time-of-flight (Q-TOF) data obtained for several tryptic peptides from standard proteins.  相似文献   

7.
The complete amino acid sequence of a novel high-potential iron-sulfur protein (HiPIP) isozyme 1 from the moderately halophilic phototrophic bacterium Ectothiorhodospira mobilis was determined by a combined approach of chemical and mass spectrometric sequencing techniques. By mass analysis of the apo- and holo-protein in the positive electrospray ionization mode using different electrospray solvents, the protein was found to be post-translationally modified by a moiety of 43 Da. Further analysis showed the nature and location of this modification to be a carbamyl group at the N-terminus of the HiPIP. This rare type of modification has previously been reported to occur in the water-soluble human lens alphaB-crystallin, class D beta-lactamases and some prokaryotic ureases, albeit at an internal lysine residue. In this paper, we discuss the mass spectrometric features of a carbamylated residue at the N-terminus of a peptide or a lysine side-chain during sequence analysis by collision-induced dissociation tandem mass spectrometry. Our data provide evidence for the first case of a prokaryotic carbamylated electron transport protein occurring in vivo.  相似文献   

8.
K Ou  T K Seow  R C Liang  S E Ong  M C Chung 《Electrophoresis》2001,22(13):2804-2811
Recently, we reported the proteome analysis of a human hepatocellular carcinoma cell line, HCC-M (Electrophoresis 2000, 21, 1787-1813), using two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). From a total of 408 unique spots excised from the 2-DE gel, 301 spots yielded good MALDI spectra. Out of these, 272 spots had matches returned from the database search leading to the identification of these proteins. Here, we report the results on the identification of the remaining 29 spots using nanoelectrospray ionization-tandem mass spectrometry (nESI-MS/MS). First, "peptide tag sequencing" was performed to obtain partial amino acid sequences of the peptides to search the SWISS-PROTand NCBI nonredundant protein databases. Spots that were still not able to find any matches from the databases were subjected to de novo peptide sequencing. The tryptic peptide sequences were used to search for homologues in the protein and nucleotide databases with the NCBI Basic Local Alignment Search Tool (BLAST), which was essential for the characterization of novel or post-translationally modified proteins. Using this approach, all the 29 spots were unambiguously identified. Among them, phosphotyrosyl phosphatase activator (PTPA), RNA-binding protein regulatory subunit, replication protein A 32 kDa subunit (RP-A) and N-acetylneuraminic acid phosphate synthase were reported to be cancer-related proteins.  相似文献   

9.
Liquid chromatography coupled with a photodiode array detector, electrospray ionization, collision‐induced dissociation and tandem mass spectrometry (LC‐DAD/ESI‐CID‐MS/MS) on a triple quadrupole (QqQ) has been used to detect and characterize polyphenols and methylxanthines in green coffee beans: three phenolic acids (caffeic acid, ferulic acid and dimethoxycinnamic acid), three isomeric caffeoylquinic acids (Mr 354), three feruloylquinic acids (Mr 368), one p‐coumaroylquinic acid (Mr 338), three dicaffeoylquinic acids (Mr 516), three feruloyl‐caffeoylquinic acids (Mr 530), four p‐coumaroyl‐caffeoylquinic acids (Mr 500), three diferuloylquinic acids (Mr 544), six dimethoxycinnamoyl‐caffeoylquinic acids (Mr 544), three dimethoxycinnamoyl‐feruloylquinic acids (Mr 558), six cinnamoyl‐amino acid conjugates, three cinnamoyl glycosides, and three methylxanthines (caffeine, theobromine and theophylline). Dimethoxycinnamic acid, three isomers of dimethoxycinnamoyl‐caffeoylquinic acids and another three of dimethoxycinnamoyl‐feruloylquinic acids, as well as the three cinnamoyl glycosides, had not previously been reported in coffee beans. Structures have been assigned on the basis of the complementary information obtained from UV‐visible spectra, relative hydrophobicity, scan mode MS spectra, and fragmentation patterns in MS2 spectra (both in the positive and negative ion modes) obtained using a QqQ at different collision energies. A structure diagnosis scheme is provided for the identification of different isomers of polyphenols and methylxanthines. Copyright © 2009 John Wiley & Sons, Ltd.  相似文献   

10.
Spectra obtained using electrospray ionization mass spectrometry (ESI-MS) of the mollusk Elysia grandifolia showed a cluster of molecular ion peaks centered at a molecular mass of 1478 Da (kahalalide F, an anticancer agent). Two new molecules, kahalalide R (m/z 1464) and S (m/z 1492) were characterized using tandem mass spectrometry. The mass differences of 14 Da suggest that they are homologous molecules. In addition, previously identified kahalalide D and kahalalide G are also reported. However, the ESI-MS of the mollusk's algal diet Bryopsis plumosa showed the presence of only kahalalide F. The amino acid sequences of kahalalide R and S are proposed using collision-induced dissociation (CID) experiments of singly and doubly charged molecular ions and by comparison with the amino acid sequence of kahalalide F. The pathway is presented for the loss of amino acid residues in kahalalide F. It is observed that there is sequential loss of amino acids in the linear peptide chain, but in the cyclic part the ring opens at the amide bond rather than at the lactone linkage, and the loss of amino acid residues is not sequential. The CID experiment of the alkali-metal-cationized molecular ions shows that the sodium and potassium ions coordinate to the amide nitrogen/oxygen in the linear peptide chain of the molecule and not to the lactone oxygen of the lactone. In the case of kahalalide D, CID of the protonated peptide opens the depsipeptide ring to form a linear peptide with acylium ion, and fragment ion signals indicate losses of amino acids in sequential order. In this study, tandem mass spectrometry has provided the detailed information required to fully characterize the new peptides.  相似文献   

11.
Three abundant small acid-soluble proteins (SASPs) from spores of Bacillus globigii were sequenced using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with post-source decay and nanoelectrospray collision-induced dissociation tandem mass spectrometry. The proteins were extracted from spores with 1 M HCl. Scanning electron micrographs of spores before and after acid extraction show that the spores retain their overall structure but have a shriveled texture following the acid treatment. Extracted SASPs were purified by high-performance liquid chromatography and molecular masses of the SASPs were identified at 7068 (SASP-1), 7332 (SASP-2), and 8889 (gamma-SASP). De novo peptide sequencing was used to determine the protein sequences. The correct ordering of peptide sequences was aided by mapping overlapping enzymatic digests and by comparison with homologous SASPs from Bacillus stearothermophilus. B. globigii is used in many field tests as a surrogate for B. anthracis. Thus complete SASP sequences from B. globigii will facilitate the development of methods for rapid identification of bacteria based on mass spectrometry and the examination of taxonomic relationships between Bacillus species.  相似文献   

12.
Electron detachment dissociation (EDD) is an emerging mass spectrometry (MS) technique for the primary structure analysis of peptides, carbohydrates, and oligonucleotides. Herein, we explore the potential of EDD for sequencing of proteins of up to 147 amino acid residues by using top-down MS. Sequence coverage ranged from 72% for Melittin, which lacks carboxylic acid functionalities, to 19% for an acidic 147-residue protein, to 12% for Ferredoxin, which showed unusual backbone fragmentation next to cysteine residues. A limiting factor for protein sequencing by EDD is the facile loss of small molecules from amino acid side chains, in particular CO(2). Based on the types of fragments observed and fragmentation patterns found, we propose detailed mechanisms for protein backbone cleavage and side chain dissociation in EDD. The insights from this study should further the development of EDD for top-down MS of acidic proteins.  相似文献   

13.
Structural studies of the high molecular mass (HMM) glutenin subunits 1Bx7 (from cvs Hereward and Galatea) and 1Bx20 (from cv. Bidi17) of bread wheat were conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). For all three proteins, MALDI-TOFMS analysis showed that the isolated fractions contained a second component with a mass about 650 Da lower than the major component. The testing and correction of the gene-derived amino acid sequences of the three proteins were performed by direct MALDI-TOFMS analysis of their tryptic peptide mixture. Analysis of the digest was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, optimizing the matrix and the acquisition parameters for each mass range. Complementary data were obtained by RP-HPLC/ESI-MS analysis of the tryptic digest. This resulted in coverage of about 98% of the sequences. In contrast to the gene-derived data, the results obtained demonstrate the insertion of the sequence QPGQGQ between Trp716 and Gln717 of subunit 1Bx7 (cv. Galatea) and a possible single amino acid substitution within the T20 peptide of subunit 1Bx20. Moreover, the mass spectrometric data demonstrated that the lower mass components present in all the fractions correspond to the major components but lack about six amino acid residues, which are probably lost from the protein C-terminus. Finally, the results obtained provide evidence for the lack of glycosylation or other post-translational modifications of these subunits.  相似文献   

14.
We report on the characteristics of the radical‐ion‐driven dissociation of a diverse array of β‐amino acids incorporated into α‐peptides, as probed by tandem electron‐capture and electron‐transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for β‐amino acids than for their α‐form counterparts. In particular, only aromatic (e.g., β‐Phe), and to a substantially lower extent, carbonyl‐containing (e.g., β‐Glu and β‐Gln) amino acid side chains, lead to N? Cβ bond cleavage in the corresponding β‐amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical‐driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α‐amino acids, ECD of peptides composed mainly of β‐amino acids reveals a shift in cleavage priority from the N? Cβ to the Cα? C bond. The incorporation of CH2 groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical‐driven β‐amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.  相似文献   

15.
金属离子对蛇毒蛋白生物活性及结构效应的影响   总被引:1,自引:0,他引:1  
从旅顺产白眉蝮蛇(Gloydius blomhoffii brevicaudus, GBB) 蛇毒中纯化得到了3种电泳和质谱纯蛋白活性组分, 其酶解肽段采用高效液相色谱-电喷雾串联质谱(HPLC-nESI-MS/MS)进行序列测定, 与其它同源性蛇毒蛋白氨基酸序列比对发现, 3种蛋白为新的蛇毒磷脂酶A2、类凝血酶和金属蛋白酶, 分别将其命名为GBB-bPLA2, GBB-TLE和GBB-MP. 电感耦合等离子体发射光谱法(ICP-AES) 测得每个GBB-bPLA2和GBB-MP中含有一个Ca2+; 每个GBB-TLE中含有2个Zn2+. Ca2+可分别使GBB-bPLA2和GBB-MP的荧光发射波长向短波方向移动2.0和1.6 nm, 使二者的荧光发射强度提高14.0%和11.0%; Ca2+的存在可显著提高二者的热稳定性, 使GBB-bPLA2和GBB-MP的热变性温度分别提高1.5和2.0 ℃. Zn2+使GBB-TLE的荧光发射强度增高4.3%, 但对GBB-TLE的酯酶水解活性、荧光发射波长和热变性温度无显著影响. 金属离子的存在能够不同程度地影响蛇毒蛋白结构热稳定性, 但对蛇毒蛋白生物活性的作用则不同.  相似文献   

16.
周艳卿  蒋小岗 《色谱》2016,34(8):752-756
Edman降解是最早建立的一种用于多肽和蛋白质氨基端测序的方法,该方法现在仍被广泛用于生物化学领域。随着高通量蛋白质组学技术的发展和应用,该方法中的异硫氰酸苯酯反应被用于修饰蛋白质氨基端,并用于检测蛋白质水解位点。但还没有异硫氰酸苯酯是否可以修饰其他氨基酸侧链并影响多肽序列分析的研究。为了探究其修饰其他氨基酸的可能性,本文利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和液相色谱-串联质谱(LC-MS/MS)研究了异硫氰酸苯酯对一个模型肽的化学修饰。质谱数据解析后发现在高浓度异硫氰酸苯酯的反应条件下,组氨酸上可以引入一个新的异硫氰酸苯酯修饰位点。这一修饰位点的发现预示着通过改变实验条件或分析方法,可以更准确地利用Edman降解和蛋白质组学技术分析多肽和蛋白质。  相似文献   

17.
The low‐abundance, low molecular weight serum proteome has high potential for the discovery of new biomarkers using mass spectrometry (MS). Because the serum proteome is large and complex, defining relative quantitative differences for a molecular species between comparison groups requires an approach with robust separation capability, high sensitivity, as well as high mass resolution. Capillary liquid chromatography (cLC)/MS provides both the necessary separation technique and the sensitivity to observe many low‐abundance peptides. Subsequent identification of potential serum peptide biomarkers observed in the cLC/MS step can in principle be accomplished by in series cLC/MS/MS without further sample preparation or additional instrumentation. In this report a novel cLC/MS/MS method for peptide sequencing is described that surpasses previously reported size limits for amino acid sequencing accomplished by collisional fragmentation using a tandem time‐of‐flight MS instrument. As a demonstration of the approach, two low‐abundance peptides with masses of ~4000–5000 Da were selected for MS/MS sequencing. The multi‐channel analyzer (MCA) was used in a novel way that allowed for summation of 120 fragmentation spectra for each of several customized collision energies, providing more thorough fragmentation coverage of each peptide with improved signal to noise. The peak list from this composite analysis was submitted to Mascot for identification. The two index peptides, 4279 Da and 5061 Da, were successfully identified. The peptides were a 39 amino acid immunoglobulin G heavy chain variable region fragment and a 47 amino acid fibrin alpha isoform C‐terminal fragment. The method described here provides the ability both to survey thousands of serum molecules and to couple that with markedly enhanced cLC/MS/MS peptide sequencing capabilities, providing a promising technique for serum biomarker discovery. Copyright © 2009 John Wiley & Sons, Ltd.  相似文献   

18.
栝楼蛋白 2: 栝楼蛋白部分化学结构的初步测定   总被引:5,自引:0,他引:5  
栝楼蛋白(Trichobitacin)是从栝楼(Trichosanthes kirilowiiMaxim, Cucurbitaceae)中新发现的核糖体失活蛋白, 分子量为27,228; pI为9.6。应用基质辅助的激光解析飞行时间质谱(MALDI-TOF-MS)和快原子轰击质谱法(FAB-MS)分别测定胰蛋白酶酶解栝楼蛋白和天花粉蛋白(Trichosanthin)的混合肽质谱, 通过比较发现了一些分子量相同的肽。由于这两种蛋白质都来源于栝楼块根, 同源性比较强, 所以这些肽序列在两种蛋白质中基本一样; 再结合蛋白N-端自动顺序仪测定栝楼蛋白N-端的结果, 确定了栝楼蛋白N-端38个氨基酸的顺序, 栝楼蛋白经胰蛋白酶酶解后所得肽段用HPLC分离纯化, 再用蛋白质自动顺序仪, DABITC/PITC双偶合手工法和质谱法共确定了栝楼蛋白N-端, C-端等100多个氨基酸残基的序列。  相似文献   

19.
Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein structure and dynamics. It has been successfully applied in the analysis of protein structure, protein folding, protein dynamics, and protein–protein and protein–DNA interactions. Using synchrotron radiolysis, exposure of proteins to a ‘white’ X‐ray beam for milliseconds provides sufficient oxidative modification to surface amino acid side chains, which can be easily detected and quantified by mass spectrometry. Thus, conformational changes in proteins or protein complexes can be examined using a time‐resolved approach, which would be a valuable method for the study of macromolecular dynamics. In this review, we describe a new application of hydroxyl radical protein footprinting to probe the time evolution of the calcium‐dependent conformational changes of gelsolin on the millisecond timescale. The data suggest a cooperative transition as multiple sites in different molecular subdomains have similar rates of conformational change. These findings demonstrate that time‐resolved protein footprinting is suitable for studies of protein dynamics that occur over periods ranging from milliseconds to seconds. In this review, we also show how the structural resolution and sensitivity of the technology can be improved as well. The hydroxyl radical varies in its reactivity to different side chains by over two orders of magnitude, thus oxidation of amino acid side chains of lower reactivity are more rarely observed in such experiments. Here we demonstrate that the selected reaction monitoring (SRM)‐based method can be utilized for quantification of oxidized species, improving the signal‐to‐noise ratio. This expansion of the set of oxidized residues of lower reactivity will improve the overall structural resolution of the technique. This approach is also suggested as a basis for developing hypothesis‐driven structural mass spectrometry experiments. Copyright © 2010 John Wiley & Sons, Ltd.  相似文献   

20.
A new methodology applicable for both high‐resolution laser desorption/ionization mass spectrometry and mass spectrometry imaging of amino acids is presented. The matrix‐assisted laser desorption ionization‐type target containing monoisotopic cationic 109Ag nanoparticles (109AgNPs) was used for rapid mass spectrometry measurements of 11 amino acids of different chemical properties. Amino acids were directly tested in 100,000‐fold concentration change conditions ranging from 100 μg/mL to 1 ng/mL which equates to 50 ng to 500 fg of amino acid per measurement spot. Limit of detection values obtained suggest that presented method/target system is among the fastest and most sensitive ones in laser mass spectrometry. Mass spectrometry imaging of spots of human blood plasma spiked with amino acids showed their surface distribution allowing optimization of quantitative measurements.  相似文献   

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