肿瘤耐药与心肌肥厚两种生物医学体系的比较蛋白质组学研究

随着人类基因组计划的完成，蛋白质组学研究已成为生命科学乃至自然科学中最活跃的学科领域之一。蛋白质组学将对我们理解后基因组学时代的基因功能有极大的帮助。差异表达蛋白质组学通过对不同样品蛋白质表达水平的比较，已经被广泛地应用在疾病的研究领域。P糖蛋白已经被证实参与介导了在急性粒白血病中发生的多药耐药，然而越来越多的研究结果表明，在急性粒白血病中多药耐药的机制是多因素的。为了寻找白血病肿瘤细胞中与耐药相关的靶蛋白，我们采用蛋白质组学研究技术，对多药耐药细胞系K562／A02的蛋白质表达谱进行了分析。 本研究采用蛋白质组学研究技术，对多药耐药细胞系K562／A02和药物敏感细胞系K562／S的总蛋白质进行双向凝胶电泳（2 DE）的差异分析，结果发现在K562／A02细胞中有41个蛋白质点差异显著，并用基质辅助激光解离飞行时间质谱（MALDI-MS）成功鉴定了大多数的蛋自点。在被鉴定出的差异蛋白质中，有一些是首次发现与耐药相关，包括chloride intracellular charmel，chaperonin，proteindisulfide-isomerase ER6o precursor and constitutive heat shock protein 70等。所得结果不但能够使我们更加深入的理解肿瘤耐药的病理机制，更重要的是通过找到肿瘤细胞发生耐药的标志性蛋白，可以为开发肿瘤耐药预测方法和寻找抗耐药肿瘤靶标新药的线索，同时也为计算机虚拟筛选设计抗耐药肿瘤新药奠定基础。除了详细的研究了K562／A02细胞系差异蛋白质组，我们还采用蛋白质组学的方法，以乳鼠心肌细胞为实验模型，研究在ARs激动的不同清况下心肌细胞蛋白质表达谱的变化。心肌肥厚是心脏工作超负荷的一种代偿反应。目前，越来越多的证据表明ARs在心肌肥厚的发生中起着非常重要的作用，但人们对其机制了解尚不全面。通过检测心肌肥厚时全蛋白表达的变化，将有助于我们全面、深入了解ARs在心肌肥厚中的作用机制。通过严格的2D胶图象分析，蛋白取点后胶内水解，高灵敏度的质谱鉴定及数据库检索，我们成功地进行了乳鼠心肌细胞肥厚为模型的蛋白质组学的研究。本实验通过比较研究不同肾上腺素受体激动剂norePinephrine伽E），phenylephxine（PE）and isoprenaline（ISO）诱导组与未诱导对照组的蛋白质组学变化，分析确定蛋白丰度差异在2倍以上的蛋白质点，并通过MALDI-MS进行蛋白鉴定。20个具有显著差异的蛋白质被鉴定，包括caspase-11，vimeniin，4-hydroxyphenylpyruvic acid dioxygenase，Modl protein，Txndcs protein等，其中一些蛋白在此之前并没发现与心肌肥厚相关，对它们在心肌肥厚中的意义和机制我们将做进一步的探讨。本研究结果将为我们更好理解肾上腺素受体之间以及肾上腺素受体与心肌肥厚之间的关系提供有益的线索，并将为最终发现可应用于临床的新的诊断和治疗心血管疾病的标志性蛋白打下基础。

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.

质谱技术在免疫分子的结构研究中的应用

质谱技术用于生物大分子的研究具有直接、简单、快速、经济等优点.近十年来,基质辅助激光解吸质谱(MALDI-MS)和电喷雾质谱(ESI-MS)在免疫学领域的研究中作出了重要贡献.本文着重对抗原、抗体、抗原-抗体复合物、抗原决定簇等免疫分子结构的质谱研究作一评述.大体分为四方面内容:免疫分子的分子量、翻译后修饰、异质性、构象变化的分析;质谱指纹图的取得和串联质谱测序;抗原-抗体复合物的证明;B-细胞表位和T-细胞表位序列的测定.这些研究结果对于理解免疫分子的免疫功能、对于疾病的早期诊断、对于发展新药和疫苗具有重要意义.

Differential proteomic analysis of neonatal cardiomyocytes in response to beta-adrenergic receptor stimulation

beta-Adrenoceptors(beta-ARs) play a critical role in regulating cardiac functions under both physiological and pathological conditions. To further explore the mechanisms through which beta-ARs perform its actions, proteomic approaches were adopted to study the global protein patterns in cultured neonatal rat cardiomyocytes exposed to isoproterenol (ISO). A modified method, "Mirror Images in One Gel", was used to improve the reproducibility and resolution power of two-dimensional electrophoresis. A 2-DE map with a good reproducibility was obtained in which 1281 70 spots were detected and about 1191 +/- 54 spots were matched, with an average matching rate of 92.9%. Nine proteins with significant changes were identified by using peptide mass fingerprinting(PMF) data obtained via MALDI-MS.

Acid effects on protein molecular weight determination

The acid effects of some proteins on measuring their molecular weights were studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry ( MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS). It was found that the signal intensity was enhanced through adjusting the acid concentration in some protein samples. In this paper, both MALDI-MS and ESI-MS was applied to examine the molecular weights of several standard proteins. And the proper acid concentration was detected in these spectra. In the meanwhile, it demonstrates that some associations of proteins in solution can be preserved into the gas phase and observed by the "soft ionization" mass spectrometry.

西洋参多糖PPQI-1～4的分离和表征

为研究生物活性西洋参多糖的性质 ,采取热水提取乙醇分级沉淀、葡聚糖凝胶分离等手段从西洋参根中分得 4个纯多糖 (PPQI- 1～ 4)。基质辅助激光解吸电离质谱 (MAL DI- MS)测定分子量 ,乙酰化衍生结合GC分析测定糖组成。甲基化结合 GC- MS分析测定糖苷键连接位点。结果表明 ,这 4个多糖化合物都是杂多糖 ,分别由不同比例的阿拉伯糖、半乳糖、葡萄糖、糖醛酸组成 ,糖醛酸含量为 30 %～ 6 0 % ,分子量范围 2～ 7万

Synthesis of monodisperse pheyl-capped oligoaniline pentamer and hexamer in the leucoemeraldine oxidation state and EB state and its UV studies

Aniline pentamer and hexamer in the leucoemeraldine oxidation state were synthesized through a novel method. The method was accomplished by the reaction of parent aniline tetramer in the pernigraniline oxidation state with diphenylamine and N-phenyl-1,4-phenylenediamine in the leucoemeraldine oxidation state respectively. The oligomers in the leucoemeraldine oxidation state were characterized by IR, NMR, elemental analysis and MALDI-MS. Aniline pentamer and hexamer in the emeraldine oxidation state were synthesized by the oxidation of Ag2O in DMF. It was found that some fragmentation occurred when the pentamer and hexamer were oxidized by (NH4)(2)S2O8 and FeCl3. 6H(2)O. The pentamer and hexamer in the emeraldine oxidation state was studied by UV/Vis spectra. The relative intensity of exciton peak for pentaaniline showed a little increase compared with that of hexaaniline.

Positive- and negative-ion matrix-assisted laser desorption/ionization mass spectrometry of saccharides

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) of oligosaccharides and polysaccharides has been investigated in detail, It is demonstrated that cationized species of oligosaccharides, [M+Na](+) and [M+K](+), are dominant products under the MALDI condition, and negative ions of oligosaccharides are not formed to any significant extent in this process, The molecular masses of polysaccharides are similarly determined by positive- and negative-ion MALDI-MS with the help of column chromatography. The distinction between positive- and negative-ion MALDI mass spectra of oligo-and polysaccharides indicates that the MALDI processes for saccharides vary with molecular mass. The matrix plays a more important role in the ionization process for oligosaccharides, while in the desorption process for polysaccharides. (C) 1998 John Wiley & Sons, Ltd.

Matrix-assisted laser desorption/ionization of ginsenosides

Molecular weight of 8 ginsenosides and the component of total saponions in American ginseng have been determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The average error of the molecular weight of each ginsenoside was found less than 0.05%. The results demonstrate that MALDI-MS is a very simple and useful method to measure the molecular weight of some high polar, thermal unstable small molecules with high sensitivity and reproducibility.

3种羧酸类基质的基质辅助激光解吸/电离质谱行为

引言基质辅助激光解吸/电离质谱(MALDI-MS)这种新的“软电离”质谱技术，能够在短短10年间，得到广泛应用与迅速发展，很大程度上要归功于基质的辅助效应。基质在样品的解吸/电离过程中，起着关键作用。对基质本身MALDI-MS的研究，不仅可以、刻认识M

Carbon nanotubes as adsorbent of solid-phase extraction and matrix for laser desorption/ionization mass spectrometry

A method with carbon nanotubes functioning both as the adsorbent of solid-phase extraction (SPE) and the matrix for matrix assisted laser desorption/ ionization mass spectrometry (MALDI-MS) to analyze small molecules in solution has been developed. In this method, 10 muL suspensions of carbon nanotubes in 50% (vol/vol) methanol were added to the sample solution to extract analytes onto surface of carbon nanotubes because of their dramatic hydrophobicity. Carbon nanotubes in solution are deposited onto the bottom of tube with centrifugation. After removing the supernatant fluid, carbon nanotubes are suspended again with dispersant and pipetted directly onto the sample target of the MALDI-MS to perform a mass spectrometric analysis. It was demonstrated by analysis of a variety of small molecules that the resolution of peaks and the efficiency of desorption/ ionization on the carbon nanotubes are better than those on the activated carbon. It is found that with the addition of glycerol and sucrose to the dispersant, the intensity, the ratio of signal to noise (S/N), and the resolution of peaks for analytes by mass spectrometry increased greatly. Compared with the previously reported method by depositing sample solution onto thin layer of carbon nanotubes, it is observed that the detection limit for analytes can be enhanced about 10 to 100 times due to solid-phase extraction of analytes in solution by carbon nanotubes. An acceptable result of simultaneously quantitative analysis of three analytes in solution has been achieved. The application in determining drugs spiked into urine has also been realized. (C) 2004 American Society for Mass Spectrometry.

A novel structural class of photoswitchable oligonucleotide

Directed Michaelis–Arbuzov reactions of support-bound internucleotide O-benzyl- or O-methyl-phosphite triesters with meta-phenylazobenzylamine or alkane-/glycol-linked a,x-diamines were effected in the presence of iodine. The corresponding tritylated phosphoramidate-linked 11-mers were fully deprotected and released from the support under standard conditions and the fast- and slow-diastereoisomers of both the E- and the Z-meta-phenylazobenzyl-appended oligomers were readily resolved by RP-HPLC. The primary amine-functionalised oligonucleotides were either purified, detritylated and then finally treated with Nhydroxysuccinimidyl carboxylic acid ester derivatives of photoswitchable moieties (Route A) or first derivatised and then subsequently purified and detritylated (Route B). This latter route enabled resolution of fast- and slow-isomers of the trityl-on oligomers bearing novel photoswitchable azopyridine or 9-alkoxyanthracene moieties using RP-HPLC, following which the pure diastereoisomers were detritylated and characterised by MALDI-MS.

Proteomic analysis reveals oxidative stress response as the main adaptative physiological mechanism in cows under different production systems

Three groups of cows representing three ranges of welfare in the production system were included in the study: two groups of Bruna dels Pirineus beef cattle maintained under different management systems (good and semiferal conditions) and a group of Alberes cows, a breed that lives in the mountains (hardest conditions).

In order to identify new stress/welfare biomarkers, serum from Bruna cows living in both environments was subjected to DIGE labelling, two-dimensional electrophoresis and MALDI-MS or ion trap MS. Identification was achieved for 15 proteins, which mainly belonged to three biological functions, the oxidative stress pathway (glutathione peroxidase (GPx) and paraoxonase (PON-1)), the acute phase protein family (Heremans Schmid glycoprotein alpha2 (α2-HSG)) and the complement system.

Biological validation included the Alberes breed. GPx and PON-1 were validated by an enzymatic assay and found to be higher and lower, respectively, in cows living in hard conditions. α2-HSG was validated by ELISA and found to be reduced in hard conditions. Other biomarkers of the redox status were also altered by living conditions: protein carbonyl content, superoxide dismutase (SOD) and glutathione reductase (GR).

Our results show that changes in the redox system are the main adaptation of cows living in challenging environmental conditions. This article is part of a Special Issue entitled: “Farm animal proteomics”.

Design, synthesis and validation of an inhibitor of CGRP degradation

Introduction: In addition to their afferent role in detection and signalling noxious stimuli, neuropeptide-containing sensory nerves may initiate and maintain chronic inflammation in diseases such as periodontitis by an efferent process known as neurogenic inflammation. Neuropeptides are susceptible to cleavage by peptidases, and therefore, the exact location and level of expression of peptidases are major determinants of neuropeptide action. Previous studies in our laboratory showed that enzyme components of gingival crevicular fluid (GCF) from periodontitis sites selectively inactivated the neuropeptide calcitonin gene-related peptide (CGRP), known to have a role in inhibiting osteoclastic bone resorption. Objectives: The aim of this study was to design and synthesise a specific inhibitor to prevent the degradation of CGRP by components of GCF. Methods: A hydroxamate-based inhibitor with a biotinylated tag was designed to ensure selectivity for CGRP and ease of use for future purification strategies. The biotinylated peptide hydroxamate contained the P1-P4 amino acid sequence of the potential CGRP cleavage site and was synthesised by solid-phase methods using standard Fmoc chemistry. Inhibition of CGRP metabolism by GCF was determined by MALDI-mass spectrometry (MALDI-MS) using pooled GCF samples from periodontitis patients as a crude source of the CGRP-degrading enzyme. Results: MALDI-MS analysis of CGRP degradation showed almost complete inhibition in the presence of the biotinylated inhibitor. Our results showed that the rate-limiting step in the cleavage of CGRP is endopeptidase cleavage, followed by carboxypeptidase attack. Conclusion: This study demonstrates that the enzyme component of GCF responsible for the degradation of CGRP can be inhibited by a biotinylated hydroxamate modelled on a potential endopeptidase cleavage site. The biotin tag on the inhibitor will facilitate our future purification of the CGRP-cleavage enzyme using a streptavidin-agarose column.