钆对费氏中华根瘤菌抑制效应的蛋白质组分析

运用双向凝胶电泳和MALDI-TOF质谱技术,分析了Gd3+对费氏中华根瘤菌USDA 205的抑制效应。结果表明,经1 mmol.L-1Gd3+处理12 h后,22个蛋白质表达量有显著变化,其中13个蛋白质表达量增加,9个蛋白质表达量下降。这些蛋白质可根据功能分为8类,包括转运蛋白、胁迫相关蛋白、代谢相关蛋白等。

Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ionization-time of flight-mass spectrometry

Peptide mass mapping analysis, utilizing a regenerable enzyme microreactor with metal-ion chelated adsorption of enzyme, combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was developed. Different procedures from the conventional approaches were adopted to immobilize the chelator onto the silica supports, that is, the metal chelating agent of iminodiacetic acid (IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the inner wall of the fused-silica capillary pretreated with NH4HF2. The metal ion of copper and subsequently enzyme was specifically adsorbed onto the surface to form the immobilized enzyme capillary microreactor, which was combined with MALDI-TOF-MS to apply for the mass mapping analysis of nL amounts of protein samples. The results revealed that the peptide mapping could routinely be generated from 0.5 pmol protein sample in 15 min at 50degreesC, even 20 fmol cytochrome c could be well digested and detected.

免疫亲和质谱法研究β_2-微球蛋白抗原表位

采用免疫亲和分离与质谱分析相结合的方法,对β2-微球蛋白抗原表位进行了系统研究.完整的抗原分子和已固定在载体(CNBr-activated Sepharose beads)上的单克隆抗体发生免疫亲和反应后,用Endoproteinase Glu-C,Trypsin,Aminopeptidase M和carboxypeptidase Y四种不同的蛋白酶依次酶解抗原分子,并采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术对与抗体连接受保护而未发生水解的肽段进行了研究.结果表明:β2-微球蛋白抗原表位位于整个蛋白分子氨基酸序列的61～67位,即为SFYLLYY.通过合成肽段的分析,证明了SFYLLYY即为抗原表位,与亲和质谱方法分析结果一致.

金属离子与钙调蛋白相互作用的基质辅助激光解析电离飞行时间质谱研究

Calmodulin is a ubiquitous calcium-binding protein in eukaryote cells and engages in various important biological pathways. In the present study, binding interactions between several metal ions and calmodulin were nvestigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).The results revealed that the specific binding of metal ions with the protein could be detected using MALDI-TOF MS.

含二硫键多肽的基质辅助激光解析电离飞行时间质谱研究

The determination of disulfide bonds becomes an important aspect of obtaining a comprehensive understanding of the chemical structure of a protein. Numerous experimental methods have been developed for the determination of disulfide bonds in proteins. Modern mass spectrometry has developed as an important tool for the analysis of disulfide bond patterns due to its advantages of being simple, rapid and sensitive. The dissociations of the disulfide bonds were detected during the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. These fragment ions were attributed to prompt fragmentation or “in-source decay” rather than “post-source decay”. For the double disulfide bonds, ions of plus sulfur and minus sulfur atoms corresponding to cleavages at different sites within the carbon-sulfur-sulfur-carbon disulfide bonds were also observed.

Investigation of Calmodulin-Peptide Interactions Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

In this report, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to study the binding interactions between calmodulin and two target peptides (melittin and substance P). Various matrix conditions were tested and the less acidic matrix DHAP and THAP were found to favor the survival of the intact calcium-calmodulin as well as the calmodulin-peptide complexes. However, the application of direct MALDI-MS to detect the intact complexes turned out to be very difficult due to the dissociation of the complexes and the formation of nonspecific aggregates. In contrast, the specific binding of the target peptides to calmodulin could be easily deduced using intensity-fading (IF) MALDI-MS. Compared with the nonbinding control, clear reduction in the ion abundances of the target peptides was observed with the addition of calmodulin.

蒙药嘎日迪五味丸中二萜生物碱的基质辅助激光解吸电离飞行时间质谱分析

采用基质辅助激光解析电离-飞行时间质谱(MALDI-TOF)对蒙药嘎日迪五味丸中的生物碱进行分析和鉴定。通过准确的相对分子质量测定,在嘎日迪五味丸中检测到了双酯型生物碱、单酯生物碱型和脂类生物碱共21种乌头生物碱,为复方中药中乌头生物碱成分的分析提供了一种新的思路。

Purification, sequencing and biological activity of polypeptide from velvet antler

The velvet antler polypeptide CNT14 was extracted and purified by gel filtration, ion exchange chromatography and RP C, which showed a single peak in HPLC chromatography and a single band in SDS-PAGE. The molecular weight measured by MALDI/TOF/MS spectrum was 1479. 9028. The polypeptide consisted mostly of Glu, Leu, Val, Pro. The amino acid sequence of the polypeptide was detected with ESI-MS/ MS, and the sequence was E-P-T-V-L-D-E-V-C-L-A-H-G-P. The experiments of biological activity of polypeptide CNT14 in vivo were carried out, and the results show that CNT14 has stimulant effects on the growth of rat HT22 cells. Then we produced the polypeptide CNT14 according the amino acid sequence by solid phase synthesis, confirmed the sequence of the polypeptide to be consistent with the amino acid sequence of polypeptide CNT14 which was separated from the velvet antler.

Lung alveolar proteomics of bronchoalveolar lavage from a pulmonary alveolar proteinosis patient using high-resolution FTICR mass spectrometry

High-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was developed and applied to the proteome analysis of bronchoalveolar lavage fluid (BALF) from a patient with pulmonary alveolar proteinosis. With use of 1-D and 2-D gel electrophoresis, surfactant protein A (SP-A) and other surfactant-related lung alveolar proteins were efficiently separated and identified by matrix-assisted laser desorption/ionization FTICR mass spectrometry . Low molecular mass BALF proteins were separated using a gradient 2-D gel. An efficient extraction/precipitation system was developed and used for the enrichment of surfactant proteins. The result of the BALF proteome analysis show the presence of several isoforms of SP-A, in which an N-non-glycosylierte form and several proline hydroxylations were identified. Furthermore, a number of protein spots were found to contain a mixture of proteins unresolved by 2-D gel electrophoresis, illustrating the feasibility of high-resolution mass spectrometry to provide identifications of proteins that remain unseparated in 2-D gels even upon extended pH gradients.

Facile synthesis and characterization of hyperbranched poly(ether amide)s generated from Michael addition polymerization of in situ created AB(2) monomers

A new straightforward strategy for synthesis of novel hyperbranched poly (ether amide)s from readily available monomers has been developed. By optimizing the reaction conditions, the AB(2)-type monomers were formed dominantly during the initial reaction stage. Without any purification, the AB(2) intermediate was subjected to further polymerization in the presence (or absence) of an initiator, to prepare the hyperbranched polymer-bearing multihydroxyl end-groups. The influence of monomer, initiator, and solvent on polymerization and the molecular weight (MW) of the resultant polymers was studied thoroughly. The MALDI-TOF MS of the polymers indicated that the polymerization proceeded in the proposed way. Analyses of H-1 NMR and C-13 NMR spectra revealed the branched structures of the polymers obtained. These polymers exhibit high-moderate MWs and broad MW distributions determined by gel permeation chromatography (GPC) in combination with triple detectors, including refractive index, light scattering, and viscosity detectors. In addition, the examination of the solution behavior of these polymers showed that the values of intrinsic viscosity [eta] and the Mark-Houwink exponent a were remarkably lower compared with their linear analogs, because of their branched nature.

基质辅助激光解吸电离飞行时间质谱表征细胞色素C

A matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF—MS) technique was used for analysis of moleculear weight of cytochrome C.The effects of three kinds of matrix,such as 2,5-dihydroxybenzoic acid(DHB),a-cyano-4-hydroxycinnamic acid(a-CHC) and sinapinic acid(SA),were used to compared and a suitable a-CHC was found.Experimental data showed that this method was properable to analysis of the congeneric biochemical samples.

Synthesis, characterization and ring-opening polymerization of cyclic (arylene phosphonate) oligomers

A series of cyclic (arylene phosphonate) oligomers were prepared by reaction of phenylphosphonic dichloride (PPD) with various bisphenols under pseudo-high dilution conditions via interfacial polycondensation. The yield of cyclic (arylene phosphonate) oligomers is over 85% by using hexadecyltrimethylammonium bromide as phase transfer catalyst (PTC) at 0 degreesC. The structures of the cyclic oligomers were confirmed by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and IR analysis. These cyclic oligomers undergo facile ring-opening polymerization in the melt by using potassium 4,4'-biphenoxide as the initiator to give linear polyphosphonate. Free-radical ring-opening polymerization of cyclic(arylene phosphonate) oligomers containing sulfur linkages was also performed in the melt using 2,2'-dithiobis(benzothiazole) (DTB) as the initiator at 270degreesC and the resulting polymer had a M-w of 8 x 10(3) with a molecular weight distribution of 4. Ring-opening copolymerization of these cyclic oligomers with cyclic carbonate oligomers was also achieved. The average molecular weight of the resulting copolymer is higher than the corresponding, homopolymer and the thermal stability of the copolymer is better than the corresponding homopolymer.

Cyclic oligomers of phenolphthalein polyarylene ether sulfone (ketone): Preparation through cyclo-depolymerisation of corresponding polymers

Cyclic oligomers of phenolphthalein polyarylene ether sulfone(ketone) were prepared through cyclo-depolymerisation of corresponding polymers using CsF as the catalyst in dipolar aprotic solvent DMAc and DMF, and a family of macrocycles containing from dimer up to at least heptamer were confirmed. by GPC, HPLC and MALDI-TOF-MS. The yields of cyclics get as high as 86.3% and 87.9% respectively.

几种无定型聚芳醚砜(酮)的成环解聚及产物的开环聚合

几种线性高分子量的无定型聚芳醚砜 (酮 )在非质子极性溶剂二甲基乙酰胺 (DMAc)和二甲基甲酰胺 (DMF)中 ,以氟化铯 (CsF)为催化剂进行解聚成环 ,所得环状低聚物由凝胶渗透色谱 (GPC)、高效液相色谱 (HPLC)和激光质谱 (MALDI TOF MS)确认 ,其中酚酞聚醚砜 (PES C)和酚酞聚醚酮 (PEK C)的解聚成环率分别高达 86.3 %和 87.9% .讨论了影响成环率的各种因素 .环状产物又在阴离子引发剂联苯双酚钾的作用下进行开环聚合重新得到高分子量的线性产物.

Synthesis and characterization of hyperbranched poly(ester-amide)s from commercially available dicarboxylic acids and multihydroxyl primary amines

A new method for syntheses of hyperbranched poly(ester-amide)s from commercially available A(2) and CBx type monomers has been developed on the basis of a series of model reactions. The aliphatic and semiaromatic hyperbranched poly(ester-amide)s with multihydroxyl end groups are prepared by in situ thermal polycondensation of intermediates obtained from dicarboxylic acids (A(2)) and multihydroxyl primary amines (CBx) in N,N-dimethylformamide. Analyses of FTIR, H-1 NMR, and C-13 NMR spectra revealed the structures of the polymers obtained. The MALDI-TOF MS of the polymers indicated that cyclization side reactions occurred during polymerization. The hyperbranched poly(ester-amide) s contain configurational isomers observed by C-13 and DEPT C-13 NMR spectroscopy. The DBs of the polymers were determined to be 0.38-0.62 by H-1 NMR or quantitive C-13 NMR and DEPT 135 spectra. These polymers exhibit moderate molecular weights, with broad distributions determined by size exclusion chromatography ( SEC), and possess excellent solubility in a variety of solvents such as N, N- dimethylacetamide, dimethyl sulfoxide, tetrahydrofuran, and ethanol, and display glass-transition temperatures (T(g)s) between -2.3 and 53.2 degrees C, determined by DSC measurements.