蛋白质结构与相互作用研究新方法——交联质谱技术

蛋白质的空间结构信息以及蛋白质间的相互作用信息对于研究蛋白质的功能有重要意义.研究蛋白质结构与相互作用的传统技术,如核磁共振技术、X射线晶体衍射技术等,对于蛋白质的纯度、结晶性和绝对量均有比较高的要求,限制了其广泛应用.交联质谱技术是近十多年来发展起来的新技术,它将质谱技术与交联技术相结合,在研究蛋白质结构与相互作用方面具有速度快、成本小、蛋白质各方面性状要求低等优势.本文就交联质谱技术各个环节的技术方法加以综述,包括交联质谱实验分离富集技术、常见交联剂特性、交联质谱数据库搜索算法、结果验证研究和交联质谱技术的应用等方面,并展望了该研究方向未来的发展.     

生物质谱技术与蛋白质组学

生物质谱技术是蛋白质组学的支撑技术.详细论述了质谱技术的分类与基本分析原理,重点论述了质谱技术的发展变化,包括基质辅助激光解吸飞行时间质谱技术,电喷雾质谱技术,MALDI-Q-TOF和MAL-DI-TOF-TOF等质谱技术,以及质谱技术在蛋白质组学研究中的应用与未来的发展和挑战.     

现代质谱技术在蛋白质组学中的应用及其最新进展

简述了蛋白质组学的概念、内容和意义,重点综述了现代质谱技术在蛋白质组学中的应用,主要包括蛋白质和肽段的鉴定和定量、蛋白质翻译后修饰的鉴定和蛋白质间相互作用的检测等。随着新的高质量精确度、分辨率、灵敏度和通量质谱仪的出现,现代质谱技术在蛋白质组学中的应用将越来越广泛,并给蛋白质组学研究带来新的机遇。     

基于生物质谱的蛋白质C末端研究进展

蛋白质的C末端在蛋白质进行各项生命活动过程中都起着极其重要的作用。它不仅标志着DNA转录翻译成蛋白质过程的初步完成,更是参与和调控了蛋白质的各种生理功能。研究蛋白质的C末端不仅有利于完整蛋白质的鉴定,对于在分子水平理解蛋白质的信号传导和生化功能是十分必要的。文中结合我们的研究工作,综述了近年来基于生物质谱的蛋白质C末端研究的相关进展,包括了C末端的识别、鉴定以及蛋白质C末端肽段富集的新方法和新技术。     

基于电感耦合等离子体质谱的蛋白质定量技术研究进展

综述了ICP-MS法应用于蛋白质定量技术方面的研究进展.蛋白质定量研究已成为蛋白质组学研究领域的热点,它是解析生物体蛋白质功能的重要途径.基于同位素标记和生物质谱分析技术是蛋白质定量最常用的方法之一,近年来,随着质谱技术的发展,电感耦合等离子体质谱(ICP-MS)技术成为元素测量的重要手段,这使其在蛋白质定量中具一定的应用前景.     

蛋白质复合体及蛋白质相互作用研究新策略—化学交联结合质谱分析法

近年来化学交联法结合质谱分析法被广泛用于蛋白质复合体结构及蛋白质相互作用的研究。研究表明这两种方法的有机结合为研究蛋白质复合体结构及蛋白质相互作用提供了一条新的途径。文章对不同类型的化学交联剂、质谱分析中的Bottom-up 与Top-down 两种研究策略,以及化学交联法结合质谱分析法在蛋白质复合体结构、蛋白质相互作用研究中的应用进行综述。这两种方法的不断发展与完善,将会极大促进生物大分子复合体结构及蛋白质相互作用的研究。     

化学交联技术在蛋白质相互作用研究中的应用

蛋白质相互作用是生命科学研究的一个重要领域.随着生物质谱的出现,利用化学交联技术研究蛋白质的相互作用已经成为切实可行的策略.文章介绍了化学交联反应的相关内容,及其在蛋白质相互作用研究中的应用,并简单探讨了甲醛作为交联剂的应用价值.     

基于质谱技术的蛋白质互作研究进展

蛋白质作为生命活动的执行者,其功能往往体现在与其他蛋白质的相互作用中,研究蛋白-蛋白相互作用对于人们深入了解和预防传染病、靶向治疗多基因疾病、阐明蛋白质的分子作用机制及各种复杂的生命现象具有重要意义。目前,有多种技术被用来研究蛋白间的相互作用,研究难点在于实时捕获瞬时或弱蛋白质间的相互作用,质谱技术（mass spectrometry, MS）可在某种程度上解决该难点。由于质谱技术可研究简单的蛋白质复合物再到大规模的蛋白质组实验,基于质谱技术研究蛋白质间相互作用被越来越多地应用于科学研究中。综述了蛋白质间相互作用检测方法的研究进展,重点介绍了氢氘交换质谱法和化学交联质谱法研究蛋白质间相互作用的优缺点及其应用,最后对基于质谱技术研究蛋白质间相互作用进行了总结与展望,以期为深入开展相关研究提供借鉴。     

生物质谱技术及其应用

质谱是带电粒子按质荷比大小顺序排列的图谱,最初主要用来测定元素或同位素的原子量,随着科学的发展及高性能质谱仪器的出现,质谱被越来越多地应用生命科学研究的许多领域,以其质辅助激光解吸附飞行时间质谱和电喷雾质谱为代表的现代生物质谱技术,为蛋白质等生物大分子的研究提供了必要的技术手段。本文在简介近年来比较常用的几种生物质谱技术的基础上,概述了生物质谱技术在蛋白质,核酸研究及检测分析等几个方面的初步应用。     

生物质谱与蛋白质组学

蛋白质组学是后基因组学时代最受关注的研究领域之一,其核心的鉴定技术——生物质谱近年来在仪器设计以及鉴定通量、分辨率和灵敏度等各方面均有质的飞跃,促进了蛋白质表达谱作图、定量蛋白质组分析、亚细胞器蛋白质组作图、蛋白质翻译后修饰以及蛋白质相互作用等蛋白质组研究各个领域的飞速发展。本综述了生物质谱技术的最新进展,及其在蛋白质组学研究中的应用。     

Chemical cross-linking and mass spectrometry to determine the subunit interaction network in a recombinant human SAGA HAT subcomplex

Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein–protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. Cross-linking with isotopically labeled BS3 d0-d4 followed by trypsin digestion allowed the identification of intra- and intercross-linked peptides using two dedicated search engines: pLink and xQuest. The identified interlinked peptides suggest a strong network of interaction between GCN5, ADA2B and ADA3 subunits; SGF29 is interacting with GCN5 and ADA3 but not with ADA2B. These restraint data were combined to molecular modeling and a low-resolution interacting model for the human SAGA HAT subcomplex could be proposed, illustrating the potential of an integrative strategy using cross-linking and mass spectrometry for addressing the structural architecture of multiprotein complexes.     

Proteomic analysis of native metabotropic glutamate receptor 5 protein complexes reveals novel molecular constituents

We used a proteomic approach to identify novel proteins that may regulate metabotropic glutamate receptor 5 (mGluR5) responses by direct or indirect protein interactions. This approach does not rely on the heterologous expression of proteins and offers the advantage of identifying protein interactions in a native environment. The mGluR5 protein was immunoprecipitated from rat brain lysates; co-immunoprecipitating proteins were analyzed by mass spectrometry and identified peptides were matched to protein databases to determine the correlating parent proteins. This proteomic approach revealed the interaction of mGluR5 with known regulatory proteins, as well as novel proteins that reflect previously unidentified molecular constituents of the mGluR5-signaling complex. Immunoblot analysis confirmed the interaction of high confidence proteins, such as phosphofurin acidic cluster sorting protein 1, microtubule-associated protein 2a and dynamin 1, as mGluR5-interacting proteins. These studies show that a proteomic approach can be used to identify candidate interacting proteins. This approach may be particularly useful for neurobiology applications where distinct protein interactions within a signaling complex can dramatically alter the outcome of the response to neurotransmitter release, or the disruption of normal protein interactions can lead to severe neurological and psychiatric disorders.     

Current trends in computational inference from mass spectrometry-based proteomics

Mass spectrometry offers a high-throughput approach to quantifying the proteome associated with a biological sample and hence has become the primary approach of proteomic analyses. Computation is tightly coupled to this advanced technological platform as a required component of not only peptide and protein identification, but quantification and functional inference, such as protein modifications and interactions. Proteomics faces several key computational challenges such as identification of proteins and peptides from tandem mass spectra as well as their quantitation. In addition, the application of proteomics to systems biology requires understanding the functional proteome, including how the dynamics of the cell change in response to protein modifications and complex interactions between biomolecules. This review presents an overview of recently developed methods and their impact on these core computational challenges currently facing proteomics.     

生物质谱在细胞信号转导研究中的应用

近几年快速发展起来的生物质谱技术 ,依靠 (酶解后肽段 )精确质量数测定和随机肽序列标签分析 ,实现了对蛋白质高通量的鉴定 ,并被成功地用于蛋白质相互作用和蛋白质磷酸化等翻译后修饰研究。与传统的研究手段相比 ,上述技术能够在一次实验中对多信号通路中所有磷酸化的蛋白质分子及其磷酸化位点进行鉴定 ,已成为蛋白质组学最新发展中令人关注的一个热点。简要综述质谱技术应用于上述工作中的 3种策略     

Chemical cross-linking and native mass spectrometry: A fruitful combination for structural biology

Mass spectrometry (MS) is becoming increasingly popular in the field of structural biology for analyzing protein three-dimensional-structures and for mapping protein–protein interactions. In this review, the specific contributions of chemical crosslinking and native MS are outlined to reveal the structural features of proteins and protein assemblies. Both strategies are illustrated based on the examples of the tetrameric tumor suppressor protein p53 and multisubunit vinculin-Arp2/3 hybrid complexes. We describe the distinct advantages and limitations of each technique and highlight synergistic effects when both techniques are combined. Integrating both methods is especially useful for characterizing large protein assemblies and for capturing transient interactions. We also point out the future directions we foresee for a combination of in vivo crosslinking and native MS for structural investigation of intact protein assemblies.     

Structural Insights into Cargo Recognition by the Yeast PTS1 Receptor

The peroxisomal matrix protein import is facilitated by cycling import receptors that shuttle between the cytosol and the peroxisomal membrane. The import receptor Pex5p mediates the import of proteins harboring a peroxisomal targeting signal of type I (PTS1). Purified recombinant Pex5p forms a dimeric complex with the PTS1-protein Pcs60p in vitro with a K_D of 0.19 μm. To analyze the structural basis for receptor-cargo recognition, the PTS1 and adjacent amino acids of Pcs60p were systematically scanned for Pex5p binding by an in vitro site-directed photo-cross-linking approach. The cross-linked binding regions of the receptor were subsequently identified by high resolution mass spectrometry. Most cross-links were found with TPR6, TPR7, as well as the 7C-loop of Pex5p. Surface plasmon resonance analysis revealed a bivalent interaction mode for Pex5p and Pcs60p. Interestingly, Pcs60p lacking its C-terminal tripeptide sequence was efficiently cross-linked to the same regions of Pex5p. The K_D value of the interaction of truncated Pcs60p and Pex5p was in the range of 7.7 μm. Isothermal titration calorimetry and surface plasmon resonance measurements revealed a monovalent binding mode for the interaction of Pex5p and Pcs60p lacking the PTS1. Our data indicate that Pcs60p contains a second contact site for its receptor Pex5p, beyond the C-terminal tripeptide. The physiological relevance of the ancillary binding region was supported by in vivo import studies. The bivalent binding mode might be explained by a two-step concept as follows: first, cargo recognition and initial tethering by the PTS1-receptor Pex5p; second, lock-in of receptor and cargo.