Conjugating oligosaccharides to proteins by squaric acid diester chemistry: rapid monitoring of the progress of conjugation, and recovery of the unused ligand

Samples that are periodically withdrawn from the mixture of a conjugation reaction can be analyzed on a picomolar scale without any work-up or pre-purification using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) in combination with the ProteinChip^® System. The technique provides rapid information about the increasing molecular mass of the glycoconjugate formed, thereby allowing termination of the process when the desired incorporation of the ligand onto the carrier protein is achieved. The excess oligosaccharide used at the onset of conjugation can be recovered and used in preparation of a similar neoglycoconjugate. The overall economy of conjugations, which often involve labor-intensive linker-equipped oligosaccharides, can be markedly increased in this way.     

广藿香根与根茎挥发油成分研究

应用气相色谱－质谱联用技术对广藿香根和根茎中挥发油成分进行了分析,从根油中分离出５０个色谱峰,鉴定了４７个化合物,占总量的９５．０２％,主要含广藿香酮（８１．７１％）、ｄ－苦橙油醇（２．８８％）、十六烷酸（２．２１％）、广藿香醇（１．９８％）和３,７,１１－三甲基－２,６,１０－十二烷三烯－２－醇乙酸酯（１．０９％）、广藿香醇（１．９８％）和３,７,１１－三甲基－２,６,１０－十二烷三烯－１－醇乙     

Optimization of a phenol extraction‐based protein preparation method amenable to downstream 2DE and MALDI‐MS based analysis of bacterial proteomes

2DE is one of the most efficient and widely used methods for resolving complex protein mixtures. For efficient analysis of complex samples, high‐resolution separation of proteins on 2D gel is essential, and for that purpose good sample preparation is crucial. In this study, we have improvized a method for preparing bacterial total cellular proteome, from a strategy applied earlier to recalcitrant plant tissues, which gave high‐quality resolution on 2DE. The method involving phenol extraction followed by methanol/ammonium acetate precipitation was first optimized for the chemolithotrophic proteobacteria Tetrathiobacter kashmirensis WT001 and Pseudaminobacter salicylatoxidans KCT001 that did not yield quality protein preps in conventional trichloroacetic acid/acetone precipitation method. Subsequently, to validate its general applicability, the method was evaluated against the trichloroacetic acid/acetone precipitation method for two other model bacteria, i.e. Escherichia coli DH5α and Mycobacterium smegmatis mc²6. Identification of at least four proteins each from the outer membrane, periplasm, and cytoplasm of T. kashmirensis by MALDI‐MS not only proved the efficiency of the method in extracting proteins from the different cellular compartments but also the amenability of the obtained protein spots toward MALDI‐MS based identification.     

Modulation of synaptic plasticity by brain estrogen in the hippocampus

The hippocampus is a center for learning and memory as well as a target of Alzheimer's disease in aged humans. Synaptic modulation by estrogen is essential to understand the molecular mechanisms of estrogen replacement therapy. Because the local synthesis of estrogen occurs in the hippocampus of both sexes, in addition to the estrogen supply from the gonads, its functions are attracting much attention.     

rhIL-12二硫键、N-糖基化位点及C端氨基酸序列分析

重组人白细胞介素12（rhIL-12）是一种已经用于治疗肿瘤,寄生虫、病毒性感染及造血障碍等疾病研究的异二聚体糖蛋白。结构确证是质量控制的重要内容,此研究对CHO细胞表达的rhIL-12二硫键配对方式、N-糖基化位点以及C端氨基酸序列进行了分析,使用Trypsin、Chymotrypsin和Glu-C三种酶分别对rhIL-12进行非还原酶解,尽可能地在其所有半胱氨酸残基之间断裂而形成二硫键相连的肽段,然后使用LC-MS/MS对酶解后的肽段样品进行分析,确定了rhIL-12样品中存在和理论配对方式相符的7对二硫键。将rhIL-12二硫键还原后并烷基化修饰保护,分别采用Trypsin,Chymotrypsin和GluC进行酶解,并用LC-MS/MS对酶解后肽段进行了质谱肽图及C端氨基酸序列分析,确定了rhIL-12 p35亚基C端氨基酸序列的8个氨基酸、p40亚基C端氨基酸序列的15个氨基酸。对rhIL-12样品还原及烷基化后用Trypsin变性酶解,所得肽段在H₂O及H₂¹⁸O水中分别用PNGase F糖苷酶处理酶切产物。并通过二级质谱分析脱糖后糖肽段分子量变化,从而确定了rhIL-12的3个N糖基化修饰位点,分别为p35亚基的71位和85位以及p40亚基的200位。通过建立酶解结合二级质谱鉴定的方法,证明了新药rhIL-12的二硫键位点、C端氨基酸序列和糖基化位点与理论一致。     

黄独微型块茎低温离体保存的GC/MS代谢组学分析

GC/MS检测方法采用初步探明黄独低温离体保存微型块茎的差异代谢物。与黄独微型块茎25℃离体保存相比较,黄独微型块茎4℃离体保存的差异性代谢物有丙氨酸（Alanine）、儿茶素（Catechin）、N,N-双（2-羟乙基）甲胺（N,N-Di-（2-Hydroxyethyl）-methanamine）、水杨酸（Salicylic acid）、柠檬酸（Citric acid）和山梨糖（Sorbose）等。在黄独微型块茎4℃离体保存中,丙氨酸（Alanine）参与氰基氨基酸代谢;儿茶素（Catechin）参与次生代谢产物生物合成、黄酮类化合物的生物合成和苯丙素的生物合成;水杨酸（Salicylic acid）参与多环芳烃降解、微生物在不同环境中的代谢、植物激素信号转导、次生代谢产物生物合成、二恶英降解、苯丙氨酸代谢、芳烃降解、植物激素生物合成、铁载体组非核糖体肽合成和苯丙素的生物合成等。柠檬酸（Citric acid）参与来自鸟氨酸、赖氨酸和烟酸的生物碱生物合成、组氨酸和嘌呤的生物碱生物合成、微生物在不同环境中的代谢、植物次生代谢产物的生物合成、2-氧代羧酸代谢、萜类和类固醇的生物合成、原核生物固碳途径、次生代谢产物生物合成、来自莽草酸途径的生物碱生物合成、来自萜类化合物和聚酮的生物碱生物合成、柠檬酸循环（TCA循环）、植物激素生物合成、乙醛酸和二羧酸代谢、双组分系统、苯丙素的生物合成以及来自鸟氨酸,赖氨酸和烟酸的生物碱生物合成等。黄独低温离体保存微型块茎差异代谢物的初步发现为进一步了解其低温离体保存的分子机制奠定了基础,也为低温离体保存黄独微型块茎的破除休眠以及其后续萌发提供了理论依据。     

Post-translational modifications in MeHg-induced neurotoxicity

Mercury (Hg) exposure remains a major public health concern due to its widespread distribution in the environment. Organic mercurials, such as MeHg, have been extensively investigated especially because of their congenital effects. In this context, studies on the molecular mechanism of MeHg-induced neurotoxicity are pivotal to the understanding of its toxic effects and the development of preventive measures. Post-translational modifications (PTMs) of proteins, such as phosphorylation, ubiquitination, and acetylation are essential for the proper function of proteins and play important roles in the regulation of cellular homeostasis. The rapid and transient nature of many PTMs allows efficient signal transduction in response to stress. This review summarizes the current knowledge of PTMs in MeHg-induced neurotoxicity, including the most commonly PTMs, as well as PTMs induced by oxidative stress and PTMs of antioxidant proteins. Though PTMs represent an important molecular mechanism for maintaining cellular homeostasis and are involved in the neurotoxic effects of MeHg, we are far from understanding the complete picture on their role, and further research is warranted to increase our knowledge of PTMs in MeHg-induced neurotoxicity.     

基于大鼠在体肠灌流模型研究白及有效部位在肠道的可吸收及代谢成分

为了考查白及有效部位在肠道的可吸收成分及其代谢特征。基于在体肠灌流模型,采用超高效液相色谱-四极杆-飞行时间质谱仪(UPLC-Q-TOF/MS)对收集到的健康SD大鼠循环肠灌流液、血清、胆汁进行分析检测,并结合对照品、质谱碎片信息和Masslynx V4.1工作站中的Single Mass Analysis功能,初步推测吸收和代谢产物的结构式。在大鼠血清和胆汁中,初步鉴定出1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸、4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯、α-异丁基苹果酸酯原型产物。在大鼠循环肠灌流液、血清和胆汁中,共鉴定出4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯的脱糖后硫酸化代谢产物和二氢菲5的葡萄糖醛酸化代谢产物,其代谢产物主要生水解和葡萄糖醛酸化反应。该方法初步探究了白及有效部位在大鼠循环肠灌流液中可吸收成分和代谢特征,为阐释白及药材的药效物质基础提供实验依据。