PTH-氨基酸的高压液相色谱分离

蛋白质和多肽是由各种氨基酸通过肽键相连而成,要阐明蛋白质的一级结构需先将其序列降解,然后测定依次降解出来的氨基酸。通常用Edman方法来降解蛋白质N-端氨基酸,使生成苯基海硫因氨基酸(简称PTH-氨基酸),而鉴定PTH-氨基酸常用的方法是气相色谱、薄层色谱和将降解生成的PTH-氨基酸再水解测定氨基酸的方法。近几年来,用高压液相色     

基于生物质谱技术的磷酰化修饰策略在多肽测序中的应用

该文建立了一种利用磷酰化修饰结合电喷雾质谱(ESI-Q-TOF)测定多肽氨基酸序列的有效方法。利用Atherton-Todd反应,以二丙基亚磷酰酯(DPP)为磷酰化试剂,应用生物质谱技术,对磷酰化修饰后的5种模型肽的磷酰化反应情况进行了系统研究,考察了磷酰化肽的二级质谱特征,并与未经磷酰化反应的肽的二级质谱特征对比。结果表明,经过磷酰化修饰后,肽的二级质谱中的a1离子信号强度明显增加,可以准确鉴定其N端氨基酸;b系列离子信息完整,信号强度增强,使得多肽C ID测序的谱图简单、清晰,有利于肽的氨基酸序列的测定;赖氨酸(K,128.10 u)和谷氨酰胺(Q,128.13 u)两种氨基酸质荷比相近,由于二者磷酰化修饰后的差异性,使其得到准确区分。经过5种已知氨基酸序列的模型肽的磷酰化后结合质谱技术进行氨基酸序列测定验证,结果表明该方法简单、快速、准确,提高了利用质谱技术进行多肽测序的准确度和灵敏度,可为蛋白质组学研究提供有效的技术手段。     

质谱技术研究海兔大脑神经节超微量多肽内切酶和酸性多肽酶解产物

采用ESI-Q-TOF质谱分析了由27个氨基酸残基组成的酸性多肽(AP)的一级结构.选用RP-HPLC和MALDI-TOF质谱非在线技术分离与鉴定海兔大脑神经节(CG)多肽与蛋白质的组成与分布,发现CG中含有AP酶解的二聚体短肽.这些短肽序列分别为2(NKDEEQRELLKAISNLL)、2(NKDEEQRELLKAISNL)、2(SGVSLLTSNKDEEQREL)和2(LTSNKDEEQRE LL),均以L-R(氨基酸残基)方式断裂.以AP为探针,结合MALDI-TOF质谱分析技术,发现CG中含有超微量的L-R多肽内切酶,其分子量为78218.25Da.本研究中的分析方法也适合于研究其他生物体内超微量多肽及多肽酶分布与功能.     

质谱技术研究海兔大脑神经节超微量多肽内切酶和酸性多肽酶解产物

采用ESI-Q-TOF质谱分析了南27个氨基酸残基组成的酸性多肽（AP）的一级结构。选用RP-HPLC和MALDI-TOF质谱非在线技术分离与鉴定海兔大脑神经节（CG）多肽与蛋白质的组成与分布，发现CG中含有AP酶解的二聚体短肽。这些短肽序列分别为2（NKDEEQRELLKAISNLL）、2（NKDEEQRELLKAISNL）、2（SGVSLLTSNKDEEQREL）和2（LTSNKDEEQRE LL），均以L-R（氨基酸残基）方式断裂。以AP为探针，结合MALDI-TOF质谱分析技术，发现CG中含有超微量的L-R多肽内切酶，其分子量为78218.25Da。本研究中的分析方法也适合于研究其他生物体内超微量多肽及多肽酶分布与功能。     

氨基酸的异硫氰酸苯酯衍生物在聚酰胺薄膜上的薄层色谱定量分析

以异硫氰酸苯酯(PITC)进行Edman降解,用于多肽和蛋白质的序列鉴定已有卅年的历史,现已有很多方法用于鉴定所得到的PTH-氨基酸衍生物。除气体色谱、高压液体色谱外,利用硅胶G的薄层色谱可以进行定性、定量分析。有的作者在聚酰胺薄膜上进行过定性分析。本文叙述了在聚酰胺薄膜上进行PTH-氨基酸薄层色谱半定量分析的方法,使用色谱扫描仪直接测出色谱点的相对面积,使定量分析工作快速、简便。     

东亚马氏钳蝎哺乳动物类神经毒素III的快原子轰击酶谱分析

应用快原子轰击酶谱分析检验了东亚马氏钳蝎哺乳动物类神经毒素III的氨基酸序列,对前人用Edman降解法测定的序列作了两处修正:(1)第60位氨基酸残基是脯氨酸而不是色氨酸;(2)C-端氨基酸是组氨酸而不是甘氨酸。分别用羧肽酶B降解和部分胰蛋白酶酶解后的质谱数据进一步证实了以上的修正。在分析中采用了化学修饰方法以减少表面抑制效应,从而得到完整的快原子轰击酶谱。     

东亚马氏钳蝎哺乳动物类神经毒素III的快原子轰击酶谱分析

应用快原子轰击酶谱分析检验了东亚马氏钳蝎哺乳动物类神经毒素III的氨基酸序列,对前人用Edman降解法测定的序列作了两处修正:(1)第60位氨基酸残基是脯氨酸而不是色氨酸;(2)C-端氨基酸是组氨酸而不是甘氨酸。分别用羧肽酶B降解和部分胰蛋白酶酶解后的质谱数据进一步证实了以上的修正。在分析中采用了化学修饰方法以减少表面抑制效应,从而得到完整的快原子轰击酶谱。     

东亚马氏钳蝎哺乳动物类神经毒素Ⅲ的快原子轰击酶谱分析

应用快原子轰击酶谱分析检验了东亚马氏钳蝎哺乳动物类神经毒素Ⅲ的氨基酸序列,对前人用 Edman 降解法测定的序列作了两处修正:(1)第60位氨基酸残基是脯氨酸而不是色氨酸;(2)(?)-端氨基酸是组氨酸而不是甘氨酸.分别用羧肽酶 B 降解和部分胰蛋白酶酶解后的质谱数据进一步证实了以上的修正.在分析中采用了化学修饰方法以减少表面抑制效应,从而得到完整的快原子轰击酶谱.     

反相高效液相色谱技术鉴定小黑麦品种

通过反相高效液相色谱(RP-HPLC)分析小黑麦种子醇溶蛋白,对供试的18个不同品种的小黑麦进行了品种鉴定和倍性水平鉴定,同时也对育种系谱相同的品种进行了系谱证实。     

蛋白质顺序测定技术(五)

(三)DABITC/PITC双偶合法~([1-13]) DABITC/PITC双偶合微量顺序测定法,是用有色试剂DABITC与多肽或蛋白质的氨基作第一次偶合,因该偶合反应产率只有25—50％左右,余下没有与DABITC偶合的氨基再用PITC作第二次偶合,由于PITC与多肽或蛋白质的偶合反应几乎是定量的,这样就保证了每个残基都反应完全,然后再裂解,转化、鉴定有色的DABTH-氨基酸。失去一个残基后的肽或蛋白质再偶合、裂解,转化,依次循环测定多肽和蛋白质顺序,其反应原理如下:     

A study of the adsorption of oxygen on silver at high pressure by electron spectroscopy

In situ X-ray photoelectron spectroscopic observation of a silver foil at 473 K exposed to oxygen at pressures up to 0.5 torr has identified three species, characterised by oxygen 1s binding energies of 528.3, 530.3 and 532.5 eV. The first two are identified as due to atomic oxygen, respectively chemisorbed at the surface and incorporated into the bulk. The third species is tentatively assigned to molecular oxygen, probably as a surface peroxidic group; both the UV photoelectron spectra and the O(1s) intensities determined at two different electron take-off angles are consistent with this interpretation.     

Pyrolsis of a flexible urethane foam

Pyrolysis of a commercial flexible urethane foam in an inert atmosphere was investigated by use of GCMS and chemical ionization mass spectroscopy. Pyrolysis products were identified and quantified at four pyrolysis temperatures. A mechanism is discussed in terms of the identified products.     

Anodization behaviour and basic property mapping in the aluminium-erbium system

A co-evaporation technique was used for depositing an Al-Er thin film combinatorial library on borosilicate glass substrates with Er concentrations between 3 and 21 at.% and a total compositional resolution of 0.25 at.% mm⁻¹. Scanning droplet cell microscopy was employed for compositional mapping of several fundamental properties of mixed oxides grown on Al-Er thin film alloys. Microstructural and crystallographic particularities of Al-Er alloys were identified along the library, and a relevant compositional threshold at around Al-5 at.% Er was identified. Further, small surface grains in the nanometre range were found at Al grain boundaries, and an initial amorphization and/or hexagonal phase nucleation was identified for Er amounts around 5 at.%. Electrochemical studies revealed the importance of this compositional threshold, as distinct maximizations of oxide formation factors and oxide electrical permittivities were observed.

     

Selective extraction of peptides in acidic human plasma by porous silica nanoparticles for peptidome analysis with 2-D LC-MS/MS

In this study, an improved method for human plasma peptidome analysis including selective porous silica nanoparticles (MCM-41) extraction and subsequent online 2-D nano-LC-MS/MS analysis was established. Enhanced enrichment efficiency for the MCM-41 extraction was obtained by adjusting the pH of the plasma sample to 2.5. A total of 1680 unique peptides were identified in the plasma sample obtained from one healthy donor, which is nearly twice the amount identified from the native state of the plasma sample. The hydrophobic property, molecular weight (MW), and pI distribution of the identified peptides at pH 2.5 and native state of the plasma sample were systematically investigated and compared. Furthermore, many unusual cleaved peptides from plasma proteins (e. g., HSA) were observed at pH 2.5, which clearly show a ladder pattern. The cleavage patterns for all of the identified peptides at pH 2.5 were summarized, and chymosin and cathepsin D were confirmed as the possible peptidases responsible for the change of cleavage pattern in peptide profiling.     

Effect of beta-lactoglobulin hydrolysis with thermolysin under denaturing temperatures on the release of bioactive peptides

In this study, bovine beta-lactoglobulin A (beta-Lg A) was hydrolysed with thermolysin under non-denaturing and heat-denaturing conditions. The peptides released during hydrolysis were identified by HPLC-MS/MS. A total of 25 peptides were identified in the hydrolysate obtained at 37 degrees C for 5 min. Some of these peptides survived to further proteolysis even at higher incubation temperatures. Furthermore, novel cleavage sites localised in the most buried zones of beta-Lg and available for thermolysin were recognised when the incubation temperature increased in the range between 60 and 80 degrees C. Three new peptides, LDA, LKPTPEGD, and LQKW, appeared after 30 min hydrolysis at these incubation temperatures, but they were not identified in the 30-min hydrolysates obtained at 37 and 50 degrees C. Of special interest was the peptide LQKW, corresponding to the fragment f(58-61) that had been previously described as a potent angiotensin-converting enzyme-inhibitor (IC50 value of 34.7 microM).     

Novel glycosylated mycosporine-like amino acids with radical scavenging activity from the cyanobacterium Nostoc commune

Mycosporine-like amino acids (MAAs) are UV absorbing pigments, and structurally distinct MAAs have been identified in taxonomically diverse organisms. Two novel MAAs were purified from the cyanobacterium Nostoc commune, and their chemical structures were characterized. An MAA with an absorption maximum at 335 nm was identified as a pentose-bound porphyra-334 derivative with a molecular mass of 478 Da. Another identified MAA had double absorption maxima at 312 and 340 nm and a molecular mass of 1,050 Da. Its unique structure consisted of two distinct chromophores of 3-aminocyclohexen-1-one and 1,3-diaminocyclohexen and two pentose and hexose sugars. These MAAs had radical scavenging activity in vitro; the 1050-Da MAA contributed approximately 27% of the total radical scavenging activities in a water extract of N. commune. These results suggest that these glycosylated MAAs have multiple roles as a UV protectant and an antioxidant relevant to anhydrobiosis in N. commune.     

The Identification of Adenosine in Cacao Products

Abstract

Adenosine has been identified as a component of defatted chocolate liquor through HPLC of aqueous extracts. The nucleoside was identified by comparing its behavior with an authentic sample in a variety of mobile phases, column types, and absorbance ratios at 254 and 245 nm.     

A two-dimensional proteome map of Shigella flexneri

Shigella flexneri is a Gram-negative facultatively intracellular pathogen responsible for bacillary dysentery in humans. In this study, extracellular proteins from the culture medium and whole cell proteins in cellular extracts of S. flexneri 2a strain 2457T were examined by two-dimensional (2-D) gel electrophoresis using immobilized pH gradient (IPG) technology. Proteins were identified by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with Mascot search program. In total, among the 488 proteins spots processed, 388 proteins were identified. The identified proteins represented 169 genes. By comparing results of Mascot search against databases of Escherichia coli and genomes of S. flexneri 2a, one S. flexneri-specific protein was identified and one possible gap was found in 2457T genome sequences. Although this proteome map is still incomplete, it is already a useful reference for future studies involving pathogenicity, vaccine development, design of novel antibacterial drugs, etc. Proteome maps and a table of all identified proteins are available on the internet at www.proteomics.com.cn.     

气相色谱-质谱联用法对6-溴-去氢甲基睾丸素合成过程中杂质的分析

采用气相色谱-质谱联用方法分析6-溴-去氢甲基睾丸素中的杂质。在30.0m×0.25mm,0.5μm的ZB-5MS毛细管柱上,柱温100℃保持3 min,后以20℃/min的速率程序升温至250℃,气化室温度270℃,载气氦气流量0.8 mL/min的条件下,6-溴-去氢甲基睾丸素及其中杂质获得很好分离。通过对各组分质谱图分析确定了6-溴-去氢甲基睾丸素中的七种杂质分别为去氢甲基睾丸素,6-烯-去氢甲基睾丸素,6-溴-17-甲基雄甾-1,4,16-三烯-3-酮的4种构象异构体以及6-溴-8-烯-去氢甲基睾丸素。     

Raman Investigation of the Low Temperature Phase Transitions in Monoclinic TlReO4

The Raman spectrum of monoclinic TlReO₄ is presented together with a factor group analysis and assignment of the bands. The phase transitions of monoclinic TlReO₄ is investigated below room temperature, and two phase transitions were identified between room temperature and 90 K. The room temperature monoclinic phase was converted to the orthorhombic phase at 170 K upon cooling, and to the tetragonal phase at 150 K. Upon heating back to room temperature the tetragonal phase persisted until the orthorhombic phase could be identified at 210 K and 220 K. From 230 K to room temperature the monoclinic phase could again be observed.