《蛋白质组学》课程教学实践与探讨

《蛋白质组学》作为一门新兴课程,以细胞内的所有蛋白质作为研究对象,系统地揭示生命的过程,为疾病的发病机制、药物的靶向机制等研究提供了新的思路。《蛋白质组学》已经在医学、药理学及微生物学等诸多领域得到了广泛的应用。在该课程教学过程中,作者尝试采用多元化的教学方法,既介绍最新的领域研究进展和新技术,又注重以承担的科研课题为例,让学生参与到具体的实验过程中来,实现理论与实践的有机结合。文中探讨了《蛋白质组学》教学思路和方法,旨在提高蛋白质组学课程的教学质量,激发学生的学习兴趣,并提高学生的创新思维能力。     

An Integrated Workflow for Proteome-Wide Off-Target Identification and Polypharmacology Drug Design

Polypharmacology,which focuses on designing drugs to target multiple receptors,has emerged as a new paradigm in drug discovery.To rationally design multi-target drugs,it is fundamental to understand protein-ligand interactions on a proteome scale.We have developed a Proteome-wide Off-target Pipeline （POP） that integrates ligand binding site analysis,protein-ligand docking,the statistical analysis of docking scores,and electrostatic potential calculations.The utility of POP is demonstrated by a case study,in which the molecular mechanism of anti-cancer effect of Nelfinavir is hypothesized.By combining structural proteome-wide off-target identification and systems biology,it is possible for us to correlate drug perturbations with clinical outcomes.     

补阳还五汤与高血压病气虚血瘀证“方证相关”的蛋白质组学研究

揭示补阳还五汤与高血压病气虚血瘀证"方证相关"的现代生物学基础,制作高血压病气虚血瘀证细胞模型。用补阳还五汤含药血清干预细胞模型,利用双向凝胶电泳(2-DE)及基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS/MS)技术筛选及鉴定差异表达的蛋白点。并对其进行生物学分析。结果高血压病气虚血瘀证组与健康对照组比较,差异蛋白质点有30个。其中,有16个蛋白上调,14个蛋白质下调。补阳还五汤组与高血压病气虚血瘀证组比较,差异蛋白质点有14个。其中,有9个蛋白上调,5个蛋白质下调。MALDI-TOF-MS/MS鉴定出:高血压病气虚血瘀证组与健康对照组差异蛋白点共有8个蛋白被成功鉴定出来。表达上调的蛋白有肽基脯氨酰异构酶A、肽基脯氨酸顺反异构酶A样1亚型、真核翻译起始因子5A-1 B亚型、微管蛋白beta-2C、CRA_b亚型、3-羟酰辅酶A脱氢酶2型异构体2。表达下调的蛋白有丙酮酸激酶同工酶M1亚型、抑制蛋白-1、抑制蛋白-1 1亚型、补阳还五汤组与高血压病气虚血瘀证组差异蛋白点共有3个蛋白被成功鉴定出来。表达上调的蛋白有丙酮酸激酶CRA_c亚型、热休克蛋白27;表达下调的蛋白有膜联蛋白A1,CRA_b亚型。这些蛋白多与促进或抑制细胞凋亡有关。说明高血压病气虚血瘀证存在着细胞凋亡,补阳还五汤可以纠正高血压病气虚血瘀证引起的细胞凋亡。这些差异蛋白可以作为高血压病气虚血瘀证的标志蛋白或补阳还五汤的作用靶点,抑制细胞凋亡可能是补阳还五汤与高血压病气虚血瘀证"方证相关"的现代生物学基础之一。     

A new chromatographic method for fast separation of active proteins

A new method with only a single chromatographic column to accomplish the separation of intact protein molecules by the combination of ion exchange chromatography and hydrophobic interaction chromatography is firstly presented. By selecting twice a suitable stationary phase, mobile phase, buffer exchange condition, and sample injection, some proteins usually required to separate with two-dimensional chromatography can now be accomplished only using the single column in one hour. In addition, the separated proteins can maintain their original three-or four-dimensional molecular structure. It would be expected that this method can also provide a new thought for sample pre-fractionation in proteomic investigation, especially, for the limitation of sample source.     

Proteomic analysis of long-term salinity stress-responsive proteins in <Emphasis Type="Italic">Thellungiella halophila</Emphasis> leaves

Salinity is one of the most severe environmental factors that may impair crop productivity. A proteomic study based on two-dimensional gel electrophoresis is performed in order to analyze the long-term salinity stress response of Thellungiella halophila, an Arabidopsis-related halophyte. Four-week-old seedlings are exposed to long-term salinity treatment. The total crude proteins are extracted from leaf blades, separated by 2-DE, stained with Coomassie Brilliant Blue, and differentially displayed spots are identified by MALDI-TOF MS or QTOF MS/MS. Among 900 protein spots reproducibly detected on each gel, 30 spots exhibit significant change and some of them are identified. The identified proteins include not only some previously characterized stress-responsive proteins such as TIR-NBS-LRR class disease resistance protein, ferritin-1, and pathogenesis-related protein 5, but also some proteins related to energy pathway, metabolism, RNA processing and protein degradation, as well as proteins with unknown functions. The possible functions of these proteins in salinity tolerance of T. halophila are discussed and it is suggested that the long-term salinity tolerance of T. halophila is achieved, at least partly, by enhancing defense system, adjusting energy and metabolic pathway and maintaining RNA structure.     

水稻叶片低温应答蛋白质组学研究进展

近年来,人们利用高通量蛋白质组学技术分析了水稻(Oryza sativa L.)叶片低温应答过程中蛋白质组的动态变化特征,在水稻叶片中鉴定到了504种低温响应蛋白质.系统分析了这些蛋白质的丰度变化模式,综述了水稻叶片应对不同程度低温胁迫(5~15℃处理0~8 d)过程中参与光合作用、糖类与能量代谢、胁迫与防御、转录与蛋白质代谢、信号转导,以及膜与转运等过程中的蛋白质丰度变化特征,为全面理解水稻低温应答的分子网络调控机制提供了线索.     

白藜芦醇诱导HepG2细胞凋亡中线粒体差异蛋白鉴定

为研究线粒体在白藜芦醇诱导人肝癌细胞系HepG2细胞凋亡中的作用机制。分离提取白藜芦醇处理的HepG2细胞及对照组细胞的线粒体蛋白质,双向电泳分离差异蛋白质,飞行时间质谱鉴定差异蛋白点,摸索并建立了一种有效分离提取细胞线粒体蛋白质和双向电泳的方法。初步分析鉴定了四个显著性差异蛋白,着丝粒蛋白Kinesin protein和CENP-E降低,证明白藜芦醇对细胞周期及细胞骨架的调节作用,Peptidase (mitochondrial processing)表达降低,线粒体核糖体蛋白L7/L12（Mitochondrial ribosomal protein L7/L12, MRP L7/L12）表达升高,表明白藜芦醇诱导HepG2细胞与其对线粒体功能的影响有关。     

蛋白质组学及其在神经科学研究中的应用

蛋白质组学是后基因组时代研究中兴起还不到十年的新型学科,但已应用到生命科学与基础医学研究中的各个领域.对蛋白质组进行大规模的分析可使我们更加深入了解生物体的结构与功能.在神经科学研究领域,对于神经突触、受体复合物以及其他的神经元和神经胶质特性的研究因为该学科的渗透,近年来取得了辉煌成就.然而,蛋白质组学相关技术用于神经系统的研究还面临着很多挑战,如脑蛋白样品的准备、脑的复杂性、有限的数据库资源和生物信息学工具等.蛋白质组学技术与其他功能基因组学研究方法,如基因芯片、网络生物学分析技术等相结合,将在基因及其功能、神经生理学与病理学之间架起一道重要的桥梁.     

植物线粒体巯基亚硝基化修饰蛋白质组学研究进展

蛋白质S-亚硝基化是一氧化氮(NO)与蛋白质半胱氨酸残基(Cys)共价连接形成S-亚硝基硫醇(-SNO)的过程,被认为是植物中体现NO生物活性的最重要途径.线粒体在依赖S-亚硝基化的NO信号转导中起关键作用.综述了应用蛋白质组学技术鉴定的植物线粒体S-亚硝基化蛋白质的特征,为认识线粒体NO调控网络体系中重要的信号与代谢通路(如光呼吸、三羧酸循环、氧化磷酸化、活性氧分子(ROS)稳态,以及蛋白质加工与周转)提供了线索.     

叶绿体巯基亚硝基化修饰蛋白质组学研究进展

蛋白质S-亚硝基化是NO与蛋白质的半胱氨酸残基共价连接形成S-亚硝基硫醇的过程.叶绿体内快速变化的氧化还原环境容易导致蛋白质S-亚硝基化修饰发生.综述了植物逆境应答过程中叶绿体蛋白质S-亚硝基化参与调节的光合电子传递、卡尔文循环、抗氧化系统、蛋白质合成、蛋白质加工与周转、Ca~(2+)介导的信号转导、氮同化、硫同化,以及四吡咯化合物合成等途径,为全面理解植物逆境应答过程的NO调控网络机制提供了线索.