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

开发 IMS-SID/MAD-MS 仪器来表征本质上无序的蛋白质结构

• 批准号: 8860745
• 负责人: Stephen Valentine
• 金额: $ 25.12万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8860745
• 关键词: Adoption; Affect; Amino Acid Sequence; Binding Sites; Buffers; Cardiovascular Diseases; Complex; Coupled; Couples; Deuterium; Development; Devices; Diabetes Mellitus; Disease; Disease Progression; Dissociation; Effectiveness; Electrospray Ionization; Exons; Foundations; Gases; Goals; Growth; Health; Huntington Disease; Huntington protein; Hybrids; Hydrogen; Individual; Ions; Knowledge; Malignant Neoplasms; Mass Spectrum Analysis; Measurable; Measurement; Methods; Modeling; Molecular; Molecular Conformation; N-terminal; NMR Spectroscopy; Neurodegenerative Disorders; Onset of illness; Outcome; Peptide Sequence Determination; Peptides; Phase; Process; Proline; Protein Conformation; Proteins; Research; Role; Site; Solutions; Spectrometry; Structure; Surface; Techniques; Technology; Testing; Time; Tube; Variant; Work; base; conformer; design and construction; flexibility; human Huntingtin protein; improved; insight; instrument; instrumentation; interest; ion mobility; mass spectrometer; novel; polyglutamine; protein aggregation; protein aminoacid sequence; protein complex; protein function; protein structure; protein structure function; prototype; public health relevance; research study; three dimensional structure; tool

 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.

 描述（由申请人提供）：我们研究的长期目标是开发强大的基于质谱（MS）的仪器和方法，用于表征生物分子的结构。特别令人感兴趣的是分子的结构确定是棘手的传统技术，如内在无序蛋白质（IDP）和大蛋白质复合物。技术进步，如这里提出的那些，为确定与从心血管疾病到癌症的各种健康状况的疾病发作和进展相关的分子机制提供了巨大的希望。 在提案期间，我们将设计和构建一个原型仪器，将新的气相分离能力与新的离子碎裂技术结合起来，用于蛋白质离子结构的表征。具体而言，将分隔的漂移管耦合到飞行时间（TOF）质谱仪。新仪器将配备表面诱导解离（SID）和亚稳态原子活化解离（MAD）功能。新仪器将使气相氢氘交换（HDX）实验与自上而下的蛋白质离子结构表征的特定构象。双片段化过程将能够根据选择的离子构象确定特定蛋白质复合物的可访问交换位点。最后，改进后的漂移管将允许在drft管设备内确定由离子激活引起的结构的这些位置。 优化的仪器将用于研究与亨廷顿（htt）蛋白相关的许多肽序列。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。具体来说，IMS-HDX-SID/MAD-MS将用于检查17个残基的N末端区域（Nt 17）、具有聚谷氨酰胺（50个残基）的N末端区域和完整外显子1模型（Nt 17 Q50 P10 KK）的结构。在此，我们将研究Nt 17上的多聚脯氨酸结合位点及其对Nt 17的类型和结构的影响 不同肽在溶液中形成的毒性低聚物种类。该仪器的成功演示不仅将提供有关与HD进展相关的分子机制的宝贵知识，而且还将为各种疾病过程的研究奠定基础。