TOP-DOWN SEQUENCING OF CROSSLINKED PROTEINS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在过去的几年中，由于新方法和更好的仪器的不断发展，质谱法已经成为蛋白质组学应用的领先技术。其中，一个分支称为结构蛋白质组学已经成长为一个强大的技术，能够提供各种各样的方法，通过结合蛋白质化学与现代质谱低分辨率表征蛋白质结构。这种方法已证明能够提供信息，可以补充那些通过高分辨率技术获得的。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，我们探索了自上而下[2，3]方法研究蛋白质相互作用和四级结构的限制和可能性。 方法：将冻干人血红蛋白（Sigma）溶解（10 μ M）于10 mM乙酸钠缓冲液（pH 7.5）中。双[磺基琥珀酰亚胺基]辛二酸酯（BS 3）的等分试样（5 mM、10 mM、25 mM、50 mM、100 mM）加入到不同的反应混合物中，并加入200- 100 mM的水溶液。在5，15，30，60，使用Advion Triversa NanoMate ESI源，用Thermo-Fisher LTQ-Orbitrap“Discovery”质谱仪分析样品。串联质谱通过LTQ-CID和HCD在C-阱或分离器-撇渣器解离（NSD）中产生，并使用Xtract软件（Thermo Scientific）去卷积。使用BUPID-Topdown（Boston University Protein Identifier-Topdown）（一种内部编写的定制编程软件算法）分析片段质量列表。 结果如下：我们分析的第一步是优化人血红蛋白和BS 3之间的交联反应和样品制备，以使混合物适合于直接质谱（MS）分析。所采用的试剂，BS 3，是一种同型双功能交联剂，含有一个胺反应性N-羟基磺基琥珀酰亚胺（NHS）酯在每一个11.4-A间隔臂的末端。为了评估反应动力学和以下的形成不同的物种，不同的反应混合物，随着交联剂的摩尔过量增加，随后进行了时间过程分析。随着反应时间的增加，样品显示出越来越高的复杂度，因此表明蛋白质修饰和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，我们能够观察到血红蛋白的α-和β-亚基的仅同源二聚体的形成，没有检测到异源二聚体。我们分离了交联的同二聚体的不同电荷状态，并结合了不同的断裂技术，LTQ-CID、C-trap中的HCD和Splele-skimmer解离（NSD），应用于不同的电荷状态，我们能够定位识别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-末端氨基与另一个亚基的K127或K139之间的共价交联。目前还没有发现可以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射线结构测定一致。它们还表明，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，而且还能够分析反应行为，以选择最低试剂量和反应时间的最佳反应条件，以保留分析中蛋白质的天然三级结构。这种方法不仅提供了蛋白质四级结构分析的可能性，即使在晶体不可用时，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，其需要更大量的样品并且对反应条件提供更低程度的控制。 1.化学交联和质谱法绘制三维蛋白质结构和蛋白质-蛋白质相互作用。A. Sinz，Mass Spectrom Rev.2006 25（4）：663-82 2.通过串联高分辨率质谱法进行的“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，McLafford FW. J Am Chem Soc 1999，121：806812 3.自上而下的方法，利用化学交联和傅立叶变换质谱法进行蛋白质结构研究。王文军.质谱技术与应用.北京：科学出版社，2003，17：155 162

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