TOP-DOWN SEQUENCING OF CROSSLINKED PROTEINS

交联蛋白质的自上而下测序

• 批准号: 7955980
• 负责人: Catherine E. Costello
• 金额: $ 3.74万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955980
• 关键词: Algorithms; Aliquot; Amines; Amino Acids; Behavior; Biology; Boston; Buffers; Charge; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Computer software; Crosslinker; Custom; Development; Dissociation; Esters; Event; Fourier Transform; Funding; Gel; Grant; Hemoglobin; Homo; Housing; Human; Institution; Kinetics; Lysine; Maps; Mass Spectrum Analysis; Medicine; Methodology; Methods; N-terminal; Post-Translational Protein Processing; Preparation; Procedures; Protein Analysis; Protein Chemistry; Protein Region; Proteins; Proteomics; Reaction; Reaction Time; Reagent; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Sampling; Signal Transduction; Sodium Acetate; Source; Structural Protein; Structure; Techniques; Technology; Tertiary Protein Structure; Time; United States National Institutes of Health; Universities; Upper arm; Work; Writing; amino group; base; bis(sulfosuccinimidyl)suberate; crosslink; dimer; instrumentation; mass spectrometer; programs; protein complex; protein crosslink; protein protein interaction; protein structure; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During the past few years mass spectrometry has emerged as leading technology for proteomics applications thanks to the continuous development of new methodologies and better instrumentation. Among these, a branch known as Structural Proteomics has grown into a powerful technique, capable of offering a wide variety of approaches to low resolution characterization of protein structure by combining protein chemistry with modern mass spectrometry. This approach has proven capable of delivering information that can be complementary to those obtained by high resolution techniques. Chemical crosslinking [1] is one of the most common methodologies employed in the analysis of protein complexes. In this work we explore limits and possibilities of a top-down [2,3] approach to the study of protein interactions and quaternary structure. Method: Lyophilized human hemoglobin (Sigma) was dissolved (10 ¿M) in 10 mM sodium acetate buffer (pH 7.5). Aliquots of Bis[sulfosuccinimidyl]suberate (BS3) (5 mM, 10 mM, 25 mM, 50 mM, 100 mM) were added to different reaction mixtures, and 200-¿l aliquots were quenched with NH4HCO3 to a final concentration of 20 mM after 5, 15, 30, 60, 120 min. Samples were analyzed as is with a Thermo-Fisher LTQ-Orbitrap "Discovery" mass spectrometer using an Advion Triversa NanoMate ESI source. Tandem mass spectra were generated by LTQ-CID and HCD in the C-trap or nozzle-skimmer dissociation (NSD) and were deconvoluted using Xtract software (Thermo Scientific). Fragment mass lists were analyzed using BUPID-Topdown (Boston University Protein Identifier-Topdown), a custom-programmed software algorithm written in-house. Results: The first step of our analysis was the optimization of the crosslinking reaction between human hemoglobin and BS3 and of the sample preparation in order to render the mixture amenable to direct mass spectrometric (MS) analysis. The reagent employed, BS3, is a homobifunctional crosslinker that contains an amine-reactive N-hydroxysulfosuccinimide (NHS) ester at each end of an 11.4-A spacer arm. In order to assess the reaction kinetics and follow the formation different species, different reaction mixtures, with increasing molar excess of crosslinking agent, were followed by a time course analysis. With increasing reaction time, the samples showed a growing degree of complexity, and thus indicated the occurrence of a variety of combinations of protein modification and both intra- and intermolecular crosslinking events, ultimately leading to the disappearance of the sample signal. Among the analyzed mixtures, we were able to observe the formation of only homo-dimers of the alpha- and beta-subunits of hemoglobin, no hetero-dimer was ever detected. We isolated different charge states of the crosslinked homodimers and with the combination of different fragmentation techniques, e.g., LTQ-CID, HCD in the C-trap and nozzle-skimmer dissociation (NSD), applied to different charge states, we were able to locate the identify the specific amino acids involved in the crosslinking reaction, and thus the region of the protein involved in the interaction. From our MS/MS experiments we were able to observe that the covalent homodimer of the beta-subunits was formed via a crosslink between the K82 residues in both chains, suggesting that, in the native tetrameric structure of hemoglobin, these two residues are located close enough in space to allow the formation of a covalent crosslink. Similar results were found for the alpha-subunits. In this case we were able to locate the covalent crosslink between the N-terminal amino group of one subunit with either the K127 or K139 of another. No fragment has yet been found that can distinguish between these two lysines. Discussion: Our results are consistent with X-ray structural determinations of hemoglobin. They also show that the well established technique of protein crosslinking for conformational analysis can be performed by a top-down methodology. With this approach we were not only able to locate the regions of protein interaction by direct analysis of protein, but also to analyze the reaction behavior in order to choose the optimal reaction conditions of lowest amount of reagent and time of reaction, in order to preserve the native tertiary structure of the protein under analysis. This methodology no only offers the possibility for quaternary structural analysis of proteins even when crystals are not available, but also expedites the normal procedures employed for chemical crosslinking, including gel based analysis that requires larger amounts of sample and offer a lesser degree of control over the reaction conditions. 1.Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions. A. Sinz, Mass Spectrom Rev. 2006 25(4):663-82 2.Top down' versus 'bottom up' protein characterization by tandem high-resolution mass spectrometry. Kelleher NL, Lin HY, Valsakovic GA, Aaserud DJ, Fridrikson EK, Beavil A, Holowka D, Gould HJ, Baird B, McLafferty FW. J Am Chem Soc 1999, 121:806812 3.A top down approach to protein structural studies using chemical cross-linking and Fourier transform mass spectrometry. Kruppa GH, Schoeninger JS, Young MM. Rapid Commun Mass Spectrom 2003, 17:155 162

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在过去的几年里，由于新的方法和更好的仪器的不断发展，质谱学已经成为蛋白质组应用的领先技术。其中，一个被称为结构蛋白质组学的分支已经发展成为一项强大的技术，能够通过将蛋白质化学与现代质谱学相结合，提供各种低分辨率表征蛋白质结构的方法。事实证明，这种方法能够提供与高分辨率技术所获得的信息相辅相成的信息。化学交联[1]是蛋白质复合体分析中最常用的方法之一。在这项工作中，我们探索了自上而下[2，3]方法研究蛋白质相互作用和四元结构的局限性和可能性。 方法：冻干人血红蛋白(Sigma)在10 mM醋酸钠缓冲液(pH 7.5)中溶解。在不同的反应混合物中加入5 mM、10 mM、25 mM、50 mM、100 mM的琥珀酸双磺基丁二亚胺(BS3)等分，5、15、30、60、120min后，用碳酸氢铵熄灭L等分至20 mM。样品按原样用Thermo-Fisher LTQ-Orbitrap“Discovery”质谱仪进行分析，质谱仪使用Advion Triversa纳米ESI源。串联质谱图由LTQ-CID和HCD在C-陷阱或喷嘴-撇脂分子解离(NSD)中产生，并用XTRACT软件(Thermo Science)解卷。使用内部编写的定制编程软件算法BUPID-TOPDOWN(波士顿大学蛋白质识别器-TOPDOWN)分析碎片质量列表。 结果：我们分析的第一步是优化了人血红蛋白与BS3之间的交联反应和样品制备，以便使混合物能够进行直接质谱分析。所使用的试剂BS3是一种同双功能交联剂，在11.4-A间隔臂的两端都含有胺反应性N-羟基磺基琥珀酰亚胺(NHS)酯。为了评价反应动力学和跟踪不同物种的形成，对不同的反应混合物，随着交联剂摩尔过量的增加，进行了时间过程分析。随着反应时间的增加，样品呈现出越来越复杂的程度，从而表明蛋白质修饰以及分子内和分子间交联事件的各种组合的发生，最终导致样品信号的消失。在分析的混合物中，我们只能观察到血红蛋白α和β亚基的同源二聚体的形成，没有检测到杂二聚体。我们分离了交联型同源二聚体的不同电荷状态，并结合不同的碎裂技术，如LTQ-CID，C-Trap中的HCD和喷嘴-撇脂分子解离(NSD)，应用于不同的电荷状态，我们能够定位和识别参与交联反应的特定氨基酸，从而识别参与相互作用的蛋白质区域。 从我们的MS/MS实验中，我们能够观察到β-亚基的共价均二聚体是通过两条链上的K82残基之间的交联形成的，这表明，在天然的血红蛋白四聚体结构中，这两个残基在空间上足够近，可以形成共价交联。阿尔法亚单位也得到了类似的结果。在这种情况下，我们能够定位一个亚基的N-末端氨基与另一个亚基的K127或K139之间的共价交联。目前还没有发现能够区分这两种赖氨酸的片段。 讨论：我们的结果与血红蛋白的X射线结构测定是一致的。他们还表明，用于构象分析的成熟的蛋白质交联技术可以通过自上而下的方法来执行。利用这种方法，我们不仅可以通过直接分析蛋白质来定位蛋白质相互作用的区域，还可以分析反应行为，以选择最低试剂用量和反应时间的最佳反应条件，从而保留被分析蛋白质的天然三级结构。这种方法不仅为蛋白质的四级结构分析提供了可能性，即使在没有晶体的情况下也是如此，而且还加快了用于化学交联的正常程序，包括基于凝胶的分析，这种分析需要更多的样品，并且对反应条件的控制程度较低。 1.化学交联和质谱学绘制蛋白质三维结构和蛋白质-蛋白质相互作用图。A·辛茨，质谱学修订版2006 25(4)：663-82 2.串联高分辨质谱仪对自上而下和自下而上蛋白质的表征。Kelleher NL，Lin HY，Valsakovic GA，Aaserud DJ，Fridrikson EK，Beavil A，Holowka D，GouldHJ，Baird B，McLafferty FW。化学学报，1999年，第121：806812 3.采用化学交联法和傅里叶变换质谱法研究蛋白质结构。鲁帕·盖尔，舍宁格，JS，年轻MM.快速质谱学2003，17：155 162