Developing IMS-SID/MAD-MS Instrumentation for Characterizing Intrinsically Disordered Protein Structure

 描述(申请人提供)：我们研究的长期目标是开发强大的基于质谱学(MS)的仪器和方法来表征生物分子的结构。特别令人感兴趣的是那些通过传统技术很难确定结构的分子，如内在无序蛋白质(IDP)和大型蛋白质复合体。这里提出的技术进步为确定从心血管疾病到癌症等各种健康疾病与疾病发生和进展相关的分子机制带来了巨大的希望。在建议的时间内，我们将设计和建造一个原型仪器，将新的气相分离能力与新的离子碎裂技术结合起来，用于表征蛋白质的离子结构。具体地说，一个分隔的漂移管将耦合到飞行时间(TOF)质谱仪。新仪器将配备表面诱导解离(SID)和亚稳态原子激活解离(MAD)能力。这台新仪器将实现气相氢氚交换(HDX)实验，对特定构象的蛋白质离子结构进行自上而下的表征。双重碎裂过程将能够根据选定的离子构象确定特定蛋白质复合体的可访问交换位置。最后，改进的漂移管将允许确定由DRFT管装置内的离子激活引起的结构的这样的位置。优化后的仪器将用于研究与亨廷顿(HTT)蛋白相关的一些肽序列。这些初步研究将测试HTT的聚脯氨酸区域在聚集过程中的作用。具体地说，IMS-HDX-SID/MAD-MS将被用于研究17个残基的N-末端区域(NT17)、带有聚谷氨酰胺的N-末端区域(50个残基)和完整的外显子1模型(Nt17Q50P10KK)的结构。在这里，我们将研究NT17上的多脯氨酸结合位点以及它对类型和结构的影响 针对不同的多肽在溶液中形成的有毒低聚物种。该仪器的成功演示不仅将提供与HD进展相关的分子机制的宝贵知识，还将为各种疾病过程的研究奠定基础。

 DESCRIPTION (provided by applicant): The long-term objective of our research is to develop powerful mass spectrometry (MS)-based instrumentation and methods for characterizing the structures of biomolecules. Of particular interest are molecules for which structure determination is intractable by conventional techniques such as intrinsically disordered proteins (IDP) and large protein complexes. Technological advances such as those proposed here hold great promise for determining molecular mechanisms associated with disease onset and progression for a diverse array of health conditions ranging from cardiovascular disease to cancer. During the proposal time period we will design and construct a prototype instrument that couples new gas-phase separation capabilities with novel ion fragmentation techniques for the characterization of protein ion structures. Specifically, a compartmentalized drift tube will be coupled to a time-of-flight (TOF) mass spectrometer. The new instrument will be outfitted with surface-induced dissociation (SID) and metastable atom activated dissociation (MAD) capabilities. The new instrument will enable gas-phase hydrogen deuterium exchange (HDX) experiments with top-down protein ion structure characterization for specific conformers. The dual fragmentation process will enable the determination of accessible exchange sites for specific protein complexes according to select ion conformations. Finally, the modified drift tube will allow the determination of such sites for structures arising from ion activation within the drft tube device. The optimized instrument will be used to study a number of peptide sequences associated with the Huntington (htt) protein. These initiatory studies will test the role of the poy- proline region of htt on the aggregation process. Specifically, IMS-HDX-SID/MAD-MS will be used to examine structures of the 17-residue N-terminal region (Nt17), the N-terminal region with poly-glutamine (50 residues), and the full exon 1 model (Nt17Q50P10KK). Here, we will investigate poly-proline binding sites on Nt17 as well as its influence on the types and structures of toxic oligomeric species formed in solution for the different peptides. The successful demonstration of the instrument will not only provide valuable knowledge regarding molecular mechanisms associated with the progression of HD but also will lay the foundation for studies of a wide variety of disease processes.