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1.
以人工合成抗菌肽1(Synthetic antimicrobial peptide 1, SAMP1)为研究模板, 采用氨基酸序列重排、 不同的带正电荷氨基酸残基和疏水性氨基酸残基取代等方法, 设计合成了8条SAMP1类似肽. 利用生物信息学软件预测了SAMP1及其类似肽的理化性质; 采用圆二色光谱(CD)技术测定其在不同环境下二级结构的变化; 采用噻唑蓝(MTT)法测定其抗菌活性; 通过红细胞溶血实验评估了这些多肽的溶血性. 结果表明, 大部分类似肽具有较低的溶血毒性和较高的广谱抗菌活性. CD光谱分析结果显示, 大部分类似肽二级结构以α螺旋和无规则卷曲为主, 在体积分数为50%的2,2,2-三氟乙醇(TFE)溶液中, α螺旋结构比例增加. 与母肽SAMP1相比, 经序列重排后得到的SAMP1-A1, SAMP1-A2和SAMP1-A3的抗菌活性变化不大, 但序列中正电荷氨基酸残基均匀分布的类似肽SAMP1-A2的溶血毒性增加. 用精氨酸(Arg)取代SAMP1序列中的赖氨酸(Lys)得到的类似肽SAMP1-A4的抗菌活性增强, 同时溶血毒性降低. 用疏水性较强的异亮氨酸(Ile)和缬氨酸(Val)取代SAMP1中的疏水性氨基酸残基, 得到的类似肽SAMP1-A5和SAMP1-A7的抗菌活性急剧降低; 用疏水性较弱的色氨酸(Trp)取代SAMP1中的疏水性氨基酸残基, 得到的类似肽SAMP1-A8的抗菌活性增强, 同时溶血毒性提高.  相似文献   

2.
爱滋病病毒中肽段的酶促合成   总被引:1,自引:0,他引:1  
陈少清  徐杰诚 《有机化学》1992,12(4):418-4284
为了进一步研究酶促合成在多肽合成中的实际应用,选择合成了爱滋病病毒(人类免疫缺损病毒,HIV-I)的gp41中氨基酸序列598-609的三个肽段,该部分是HIV-I中的2个抗原决定簇部分,H-Leu-Glg-Leu-Trp-Glg-cgs-Ser-Glg-Lgs-Leu-Ile-Cgs-OH可以作为抗原来检测HIV抗体.  相似文献   

3.
An evaluation has been made of various strategies for obtaining internal amino acid sequence data from electrophoretically separated proteins. Electroblotting, in situ proteolysis and extraction, and direct electroelution are compared. Electroblotting of protein or peptides from gels resulted in poor yields (typically, 1-7%). However, higher yields (3-67%) were achieved by in situ enzymatic cleavage followed by acid extraction of the peptides from the gel. Peptides extracted from the gel were separated by reversed-phase high-performance liquid chromatography (RP-HPLC), on short, small-bore columns (100 x 2.1 mm I.D.), to enable recovery of peptides in small volumes (ca. 50 microliters) suitable for microsequence analysis. Capillary zone electrophoresis under acidic conditions (pH 2.5) was used to assess peptide purity before sequence analysis. Cysteine residues were identified in unmodified proteins or peptides by a characteristic phenylthiohydantoin (PTH)-amino acid derivative during sequence analysis. This derivative does not co-chromatograph with any known PTH-amino acid. Direct electrophoretic elution of protein from gels yielded between 45-50% of applied protein. Proteins recovered from gels by electrophoretic elution required further purification by inverse-gradient RP-HPLC [R. J. Simpson, R. L. Moritz, E. C. Nice and B. Grego, Eur. J. Biochem., 165 (1987) 21] to remove sodium dodecylsulphate and acrylamide-related contaminants for sequence analysis.  相似文献   

4.
[structures: see text] A simple and highly efficient Fmoc solid-phase protocol for synthesizing the antimicrobial decapeptide gramicidin S and various labeled analogues is presented. When preparing the linear precursor peptides (1a-e), a systematic permutation of the starting amino acid within the cyclic sequence gave different yields between 51% and 93%. Also the subsequent step of cyclization gave widely diverging yields between 26% and 74%, depending again on the starting amino acid. The ease of cyclization was found to correlate with the tendency of the respective linear precursor peptide to assume a preorganized conformation, as observed by circular dichroism. The overall yield is thus critically dependent on the starting amino acid and can be raised from 20% to 70% using (D)Phe. The choice of coupling agent and its counterion was found to play only a marginal role. Irrespective of being able to assume a preorganized conformation, none of the linear precursor peptides exhibited any antimicrobial or hemolytic activity. Using the optimized protocol, which involves only simple Fmoc-couplings and requires no intermittent purification steps, several gramicidin S analogues (3-8) containing 19F-labeled phenylglycine derivatives and/or 15N-labeled amino acids were synthesized for solid-state NMR structure analysis.  相似文献   

5.
A new complex of cyclic peptide lactone antibiotics from Bacillus subtilis, which we named maltacines, has recently been described. The structure elucidation of four of them is reported in this paper. The amino acid sequences and structures of the peptides were found by MSn of the ring-opened linear peptides that gave uninterrupted sequences of Bn and Y'n ions. The identities of three unknown residues in the sequences were solved by a combination of derivatization with phenyl isothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange. The nature and position of the cyclic structure were revealed by a chemoselective ring opening with Na18OH and was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. For verification of the structure of the B2+ ion, peptides with different combinations of P/Q and P/K at the N-terminus were synthesized. The structures of the four peptides were found to be as follows: B1a/B2a, cyclo-4,12(P-Q-Y-HNLeu-A-E-T-Y-Orn-103-Y-I-OH); and B1b/B2b, cyclo-4,12(P-Q-Y-HNLeu-A-E-T-Y-K-103-Y-I-OH).  相似文献   

6.
Peptide sequencing is critical to the quality control of peptide drugs and functional studies of active peptides. A combination of peptidase digestion and mass spectrometry technology is common for peptide sequencing. However, such methods often cannot obtain the complete sequence of a peptide due to insufficient amino acid sequence information. Here, we developed a method of generating full peptide ladders and comparing their MS2 spectral similarities. The peptide ladders, of which each component was different from the next component with one residue, were generated by continuous digestion by peptidase (carboxypeptidase Y and aminopeptidase). Then, based on the characteristics of peptide ladders, complete sequencing was realized by comparing MS2 spectral similarity of the generated peptide ladders. The complete amino acid sequences of bivalirudin, adrenocorticotropic hormone, and oxytocin were determined with high accuracy. This approach is beneficial to the quality control of drug peptides as well as the identification of novel bioactive peptides.  相似文献   

7.
We describe CHASE, a novel algorithm for automated de novo sequencing based on the mass spectrometric (MS) fragmentation analysis of tryptic peptides. This algorithm is used for protein identification from sequence similarity criteria and consists of four steps: (1) derivatization of tryptic peptides at the N-terminus with a negatively charged reagent; (2) post-source decay (PSD) fragmentation analysis of peptides; (3) interpretation of the mass peaks with the CHASE algorithm and reconstruction of the amino acid sequence; (4) transfer of these data to software for protein identifications based on sequence homology (Basic Local Alignment Search Tool, BLAST). This procedure deduced the correct amino acid sequence of tryptic peptide samples and also was able to deduce the correct sequence from difficult mass patterns and identify the amino acid sequence. This allows complete automation of the process starting from MS fragmentation of complex peptide mixtures at low concentration (e.g. from silver-stained gel bands) to identification of the protein. We also show that if PSD data are collected in a single spectrum (instead of the segmented mode offered by conventional matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrumentation), the complete workflow from MS-PSD data acquisition to similarity-based identification can be completely automated. This strategy may be applied to proteomic studies for protein identification based on automated de novo sequencing instead of MS or tandem MS patterns. We describe the Charge Assisted Sequencing Engine (CHASE) algorithm, the working protocol, the performance of the algorithm on spectra from MALDI-TOFMS and the data comparison between a TOF and a TOF-TOF instrument.  相似文献   

8.
设计合成了一类侧链带有络合基团的非天然氨基酸, 即侧链带有N,N-二羧甲基氨甲基、N,N-二酰胺甲基氨甲基和N,N-二羟乙基氨甲基的苯丙氨酸衍生物, 并将这类非天然氨基酸用于促性腺激素释放激素(LHRH)类似物的固相合成. 高效液相色谱分析结果表明, 粗肽的纯度较好, 易于纯化; 用电喷雾质谱测定了多肽的分子量. 这些非天然氨基酸可作为其它肽类药物合成的构建单元.  相似文献   

9.
10.
Strong non-covalent interactions such as biotin-avidin affinity play critical roles in protein/peptide purification. A new type of 'fluorous' (fluorinated alkyl) affinity approach has gained popularity due especially to its low level of non-specific binding to proteins/peptides. We have developed a novel water-soluble fluorous labeling reagent that is reactive (via an active sulfo-N-hydroxylsuccinimidyl ester group) to primary amine groups in proteins/peptides. After fluorous affinity purification, the bulky fluorous tag moiety and the long oligoethylene glycol (OEG) spacer of this labeling reagent can be trimmed via the cleavage of an acid labile linker. Upon collision-induced dissociation, the labeled peptide ion yields a characteristic fragment that can be retrieved from the residual portion of the fluorous affinity tag, and this fragment ion can serve as a marker to indicate that the relevant peptide has been successfully labeled. As a proof of principle, the newly synthesized fluorous labeling reagent was evaluated for peptide/protein labeling ability in phosphate-buffered saline (PBS). Results show that both the aqueous environment protein/peptide labeling and the affinity enrichment/separation process were highly efficient.  相似文献   

11.
A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins.  相似文献   

12.
The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.  相似文献   

13.
Polystyrene resins cross-linked with di(ethylene glycol) dimethacrylate (DEGDMA) and tri(ethylene glycol) dimethacrylate (TEGDMA), DEGDMA-PS and TEGDMA-PS, were synthesized by suspension copolymerization. Four functionalized resins, chloromethyl resin, 4-hydroxymethylphenoxymethyl resin (Wang resin), 4-methylbenzhydrylamine resin (MBHA resin) and 2-chlorotrityl chloride resin, were prepared from DEGDMA-PS and TEGDMA-PS. DEGDMA-PS and TEGDMA-PS showed high reactivity in the functionalization reactions in comparison with Merrifield resin (polystyrene cross-linked with divinylbenzene, DVB-PS). DEGDMA-PS-Wang resin and TEGDMA-PS-Wang resin were used as the solid-phase support for the synthesis of a difficult sequence, the fragment of acyl carrier protein 65-74. The yields of the crude peptide synthesized using DEGDMA-PS-Wang resin, TEGDMA-PS-Wang resin and DVB-PS-Wang resin were 92.3%, 91.6% and 78.8%, respectively. The purities of the crude peptides were 85.7%, 88.1% and 73.3%, respectively.  相似文献   

14.
A new complex of cyclic peptide lactone antibiotics from Bacillus subtilis, which we named Maltacines has recently been described. The structure elucidation of three of them is reported in this paper. The amino acid sequences and structures of the peptides were found by MS(n) of the ring-opened linear peptides that gave uninterrupted sequences of Bn and Y'n ions. The identities of four unknown residues in the sequences were solved by a combination of derivatisation with phenylisothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange. The nature and position of the cyclic structure was disclosed by a chemo-selective ring opening with Na18OH and was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. For verification of the structure of the B2 + ion, peptides with different combinations of P/Q and P/K at the N-terminus were synthesized. The structures of the four peptides is tentatively suggested to be: D1a: cyclo(4,12)-P-Q-Y-Adip-A-E-T-Y-Orn-HGly-Y-I-OH, D1b: cyclo(4,12)-P-Q-Y-Adip-A-E-T-Y-Orn-S-Y-I-OH and D1c: cyclo(4,12)-P-Q-Y-Adip-A-E-T-Y-K-S-Y-I-OH. Adip = aminodihydroxy pentanoic acid and HGly = hydroxyglycine.  相似文献   

15.
肿瘤细胞表面的抗原多肽能够被细胞毒T淋巴细胞特异性识别而引起免疫应答,因此有可能用于研制基于多肽的抗肿瘤疫苗。用弱酸将人肝癌细胞系HLE细胞表面抗原多肽和人正常肝细胞表面多肽洗脱后,经RP-HPLC分离,选择HLE细胞表面特异性多肽进行纳升电喷雾串联质谱(nanoESI-MS/MS)测序,共测定5个色谱峰中的20个多肽序列,分子量分布范围为1000~2000 Da。借助M asSeq软件分析出其中12个多肽的序列。经数据库查寻,其中的3个肽段分别来自钙调节蛋白、核蛋白S19和伴侣蛋白10。这些多肽的生物学功能及与肿瘤的关系值得深入研究。该研究表明nanoESI-MS/MS是测定微量混合多肽序列的最有效方法。  相似文献   

16.
The cyclization of linear peptides from six to nine amino acids in length and containing between two and four pseudoproline turn inducers derived from serine or threonine was investigated to determine the effect of peptide length, amino acid composition and spacing between the pseudoproline residues on macrocyclization yield.  相似文献   

17.
A computer program (COMPOST) is described that carries out predictive computations on known amino acid sequences. The program is designed to be of use to mass spectrometrists with an interest in protein and peptide sequencing. Mass values (monoisotopic and average) for protonated peptide and protein molecules and elemental compositions are calculated. COMPOST also calculates mass to charge ratio values for protonated peptides expected from specified digests, locates specified amino acid subsequences or peptides of a specifIed molecular weight within a longer sequence, and predicts mass to charge ratio values for fragment ions from high-energy collision-induced dissociation of protonated peptides.  相似文献   

18.
血红蛋白片段的合成及生物活性   总被引:1,自引:0,他引:1  
采用多肽固相合成方法, 以Wang 树脂为载体, Fmoc为N-端氨基酸保护基, HOBt-HBTU为缩合试剂, 合成了一系列血红蛋白α链的片段, 产物经RP-HPLC和质谱进行了确定. 生物活性研究结果表明, 该系列多肽具有较高的血管紧张素Ⅰ转换酶抑制活性, 但不具有α-葡萄糖苷酶抑制活性.  相似文献   

19.
Little data exist on the structure and function of compressible elastomeric proteins such as abductin. An understanding of the underlying structural features of these proteins may lead to the development of a new class of highly tailored "compressible" hydrogels. To that effect, in this work, the structure of abductin was investigated by means of studies on several synthetic peptides corresponding to the most frequent sequences of abductin. In particular, the 10 amino acid abductin peptide sequence FGGMGGGNAG, tandem repeated in the protein, and two related 25 and 40 amino acid polypeptides were synthesized. These peptides were studied with regard to secondary structure, self-assembly, and polymer morphology. The results obtained with these peptides allow us to propose a preliminary structure-elasticity relationship for abductin not dissimilar from that currently accepted for elastin.A possible mechanism of elasticity relating abductin to elastin.  相似文献   

20.
An understanding of the gas-phase dissociation of protonated peptides within the mass spectrometer is essential for automated high-throughput protein identification. In this communication we describe a facile cleavage of the Gln-Gly peptide bond under low-collisional energy conditions. A variety of synthetic peptides have been analysed where key amino acids have been substituted within the sequence PQGPPQQGGR, which is a consensus repeat present in the tryptic peptides of acidic proline-rich protein 1 (PRP-1). The collision-induced dissociation spectra obtained from the PRP-1 tryptic peptides and the synthetic peptides indicate that facile Gln-Gly cleavage occurs when an X-Gln-Gly-Y sequence is present in a peptide, where X is any amino acid and Y any amino acid other than Gly.  相似文献   

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