TOP-DOWN SEQUENCING OF CROSSLINKED PROTEINS

交联蛋白质的自上而下测序

• 批准号: 8170937
• 负责人: Catherine E. Costello
• 金额: $ 3.67万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170937
• 关键词: Algorithms; Aliquot; Amines; Amino Acids; Behavior; 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; Methodology; Methods; N-terminal; Post-Translational Protein Processing; Prealbumin; Preparation; Procedures; Protein Analysis; Protein Chemistry; Protein Region; Proteins; Proteomics; Reaction; Reaction Time; Reagent; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Sampling; Signal Transduction; Site; Sodium Acetate; Source; Structural Protein; Structure; Techniques; Technology; Tertiary Protein Structure; Time; United States National Institutes of Health; Universities; Work; Writing; amino group; arm; 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. 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. 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. We have now extended the study to include the tetrameric protein transthyretin, and have again succeeded in finding crosslinking sites. To obtain the best results, these experiments are now being carried out on the newly installed Bruker 12-T FTMS. In addition, chromatographic methods for enriching the crosslinked products have been developed. 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

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 在过去的几年中，由于新方法和更好仪器的不断发展，质谱分析已成为蛋白质组学应用的领先技术。其中，被称为结构蛋白质组学的一个分支已经发展成为一种强大的技术，能够通过将蛋白质化学与现代质谱相结合，提供多种方法来低分辨率表征蛋白质结构。事实证明，这种方法能够提供与通过高分辨率技术获得的信息相补充的信息。化学交联 [1] 是蛋白质复合物分析中最常用的方法之一。在这项工作中，我们探讨了自上而下 [2,3] 方法研究蛋白质相互作用和四级​​结构的局限性和可能性。 方法：将冻干人血红蛋白 (Sigma) 溶解 (10 µM) 于 10 mM 乙酸钠缓冲液 (pH 7.5) 中。将等份双[磺基琥珀酰亚胺基]辛二酸酯(BS3)(5 mM、10 mM、25 mM、50 mM、100 mM)添加到不同的反应混合物中，并在5、15、30、60、120分钟后用NH 4 HCO 3 淬灭200-2等份至20 mM的终浓度。使用 Thermo-Fisher LTQ-Orbitrap“Discovery”质谱仪使用 Advion Triversa NanoMate ESI 源对样品进行分析。串联质谱由 C-trap 或喷嘴撇渣器解离 (NSD) 中的 LTQ-CID 和 HCD 生成，并使用 Xtract 软件（Thermo Scientific）解卷积。使用 BUPID-Topdown（波士顿大学蛋白质标识符-Topdown）（一种内部编写的定制编程软件算法）对片段质量列表进行分析。 我们分析的第一步是优化人血红蛋白和 BS3 之间的交联反应以及样品制备，以使混合物适合直接质谱 (MS) 分析。所使用的试剂 BS3 是一种同双功能交联剂，在 11.4-A 间隔臂的两端均含有胺反应性 N-羟基磺基琥珀酰亚胺 (NHS) 酯。为了评估反应动力学并跟踪不同物质、不同反应混合物的形成，随着交联剂摩尔过量的增加，进行时程分析。随着反应时间的增加，样品表现出越来越复杂的程度，从而表明发生了多种蛋白质修饰组合以及分子内和分子间交联事件，最终导致样品信号消失。在分析的混合物中，我们能够观察到仅形成血红蛋白α和β亚基的同二聚体，没有检测到异二聚体。我们分离了交联同源二聚体的不同电荷状态，并结合不同的片段化技术，例如LTQ-CID、C-trap中的HCD和喷嘴撇渣器解离（NSD），应用于不同的电荷状态，我们能够确定参与交联反应的特定氨基酸，从而确定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亚基的共价同二聚体是通过两条链中的K82残基之间的交联形成的，这表明，在血红蛋白的天然四聚体结构中，这两个残基在空间上足够接近以允许形成共价交联。对于α亚基也发现了类似的结果。在这种情况下，我们能够定位一个亚基的 N 端氨基与另一个亚基的 K127 或 K139 之间的共价交联。尚未发现可以区分这两种赖氨酸的片段。 我们的结果与血红蛋白的 X 射线结构测定一致。他们还表明，用于构象分析的成熟蛋白质交联技术可以通过自上而下的方法进行。通过这种方法，我们不仅能够通过直接分析蛋白质来定位蛋白质相互作用的区域，还能够分析反应行为，以选择最低试剂用量和反应时间的最佳反应条件，从而保留所分析蛋白质的天然三级结构。这种方法不仅提供了即使在没有晶体的情况下也可以进行蛋白质四级结构分析的可能性，而且还加快了化学交联所采用的正常程序，包括基于凝胶的分析，该分析需要大量的样品，并且对反应条件的控制程度较低。 我们现在将研究范围扩大到四聚体蛋白转甲状腺素蛋白，并再次成功找到了交联位点。为了获得最佳结果，这些实验现在正在新安装的 Bruker 12-T FTMS 上进行。此外，还开发了富集交联产物的色谱方法。 1.化学交联和质谱绘制三维蛋白质结构和蛋白质-蛋白质相互作用。 A. Sinz，质谱修订版 2006 25(4):663-82 2.通过串联高分辨率质谱法进行“自上而下”与“自下而上”蛋白质表征。 Kelleher NL、Lin HY、Valsakovic GA、Aaserud DJ、Fridrikson EK、Beavil A、Holowka D、Gould HJ、Baird B、McLafferty FW。美国化学学会杂志 1999，121：806 812 3.使用化学交联和傅里叶变换质谱法进行蛋白质结构研究的自上而下的方法。 Kruppa GH、Schoeninger JS、Young MM。快速通讯质谱 2003 年，17:155 162