TOP-DOWN SEQUENCING OF CROSSLINKED PROTEINS

交联蛋白质的自上而下测序

• 批准号: 8365563
• 负责人: Catherine E. Costello
• 金额: $ 3.38万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365563
• 关键词: Algorithms; Aliquot; Amino Acids; Behavior; Biology; Boston; Buffers; Charge; Chemicals; Computer software; Custom; Development; Dissociation; Event; Fourier Transform; Funding; Gel; Grant; Hemoglobin; Homo; Housing; Human; Kinetics; Maps; Mass Spectrum Analysis; Medicine; Methodology; Methods; National Center for Research Resources; Organized by Structure Protein; Post-Translational Protein Processing; Prealbumin; Principal Investigator; Procedures; Protein Analysis; Protein Chemistry; Protein Region; Proteins; Proteomics; Reaction; Reaction Time; Recombinants; Research; Research Infrastructure; 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; base; bis(sulfosuccinimidyl)suberate; cost; crosslink; dimer; instrument; 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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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. Lyophilized human hemoglobin or recombinant transthyretin was dissolved in 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 the reactions were quenched with NH4HCO3 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 or the Bruker SolariX 12-T FTMS. Tandem mass spectra were generated by LTQ-CID and HCD in the C-trap or nozzle-skimmer dissociation (NSD) on the LTQ-Orbitrap or by SORI-CID , ECT or ETD on the SolariX and were deconvoluted using the proprietary software for each instrument. Fragment mass lists were analyzed using BUPID-Topdown (Boston University Protein Identifier-Topdown), a custom-programmed software algorithm written in-house. 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, 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 interactions. Our results are consistent with X-ray structural determinations of hemoglobin and TTR. 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 have been able to locate the regions of protein interaction by direct analysis of protein, and to analyze the reaction behavior in order to choose the optimal reaction conditions, 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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 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。 冻干人血红蛋白或重组转甲状腺素在pH 7.5的醋酸钠缓冲液中溶解。将5 mM、10 mM、25 mM、50 mM、100 mM等分的磺基琥珀酸双酯(BS3)加入不同的反应混合物中，在5、15、30、60、120min后用NH4HCO3熄灭反应。样品用Thermo-Fisher LTQ-Orbitrap“Discovery”质谱仪进行分析，质谱仪使用Advion Triversa Nanomate ESI源或Bruker SolariX 12-T FTMS。串联质谱由LTQ-Orbitrap上的C-Trap或喷嘴-撇油解离(NSD)中的LTQ-CID和HCD或SolariX上的Sori-CID、ECT或ETD产生，并使用每个仪器的专有软件进行解卷。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。 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态，并结合不同的碎裂技术，应用于不同的电荷态，我们能够定位和鉴定参与交联化反应的特定氨基酸，从而识别参与相互作用的蛋白质区域。 我们的结果与血红蛋白和TTR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，我们可以通过直接分析蛋白质来定位蛋白质相互作用的区域，并分析反应行为，以选择最佳反应条件，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。 我们现在已经将这项研究扩展到包括四聚体蛋白转甲状腺素，并再次成功地找到了交联点。为了获得最好的结果，这些实验现在正在新安装的布鲁克12-T FTM上进行。此外，还开发了富集交联物的层析方法。 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