INSTRUMENTATION FOR ANALYSIS OF HIGH MASS BIOMOLECULES

描述(申请人提供)：这项研究的总体主旨是开发新的和改进的方法来操纵和询问大分子离子，重点放在多肽种类上，包括来自酶或化学消化的多肽、整个蛋白质和蛋白质复合体。最终目标是最大限度地从感兴趣的蛋白质和蛋白质复合体中获得结构信息，以便识别和表征它们。这项工作的重点是离子形成，离子化学，以及与质谱学和串联质谱学相关的仪器。气相离子可作为目标凝聚相物种的替代物。串联质谱仪中的结构信息来源于离子化学和对带电反应产物质量的准确测量。离子的性质(例如，质子化分子与自由基阳离子)在其化学中起主要作用，反应条件也是如此。这项研究的前提是，新的离子化学、新的工具和新的方法可以解决目前质谱学从大的多肽物种中获得结构信息的能力方面的许多限制。它还基于这样的前提，即将电离过程与离子操纵/询问和质量分析步骤分开是可取的，也是可能的，以便每个步骤都可以单独优化。为了进一步增加从质谱学和串联质谱学获得的蛋白质结构信息，我们提出了以下具体目标：具体目标1：基于离子化学的新的和改进的蛋白质鉴定和结构表征方法。具体目标2：用于“混合”串联质谱仪中肽和蛋白质的串联质谱学的新仪器和方法。具体目标3：发展通过基质辅助激光解吸电离(MALDI)电离的蛋白质串联质谱学的新方法。这些目标将通过开发用于离子转化的新的离子/离子反应、扩大离子激活方法的范围、开发支持新的离子化学的仪器以及开发由于离子化学和仪器的进步而能够识别/表征蛋白质的新方法来实现。公共卫生相关性：该项目旨在改进蛋白质识别/表征。识别蛋白质并确定其翻译后修饰(即修饰的身份和位置)的能力是许多基础生物医学研究的基础。这一能力也是识别蛋白质生物标记物及其与疾病相关性的关键。该项目试图通过极大地扩大通过质谱学获得的信息内容来解决这一任务。

DESCRIPTION (provided by applicant): The overall thrust of this research initiative is to develop new and improved means for manipulating and interrogating macro-molecular ions with strong emphasis on polypeptide species, including peptides derived from enzymatic or chemical digestion, whole proteins, and protein complexes. The ultimate objective is to maximize the structural information that can be derived from proteins and protein complexes of interest for the purpose of their identification and characterization. The work involves emphases in ion formation, ion chemistry, and instrumentation pertinent to mass spectrometry and tandem mass spectrometry. Gas-phase ions serve as surrogates for the condensed-phase species of interest. Structural information in tandem mass spectrometry is derived both from ion chemistry and the accurate measurement of the masses of the charged reaction products. The nature of the ion (e.g., protonated molecule versus radical cation) plays a major role in its chemistry, as do the reaction conditions. This research is based on the premise that new ion chemistries, new tools, and new methodologies can address many of the current limitations in the ability of mass spectrometry to derive structural information from large polypeptide species. It is also based on the premise that it is both desirable and possible to separate the ionization process from the ion manipulation/interrogation and mass analysis steps so that each can be optimized individually. To further increase the protein structural information accessible from mass spectrometry and tandem mass spectrometry, we propose the following specific aims: Specific Aim 1: New and improved ion chemistry-based approaches for protein identification and structural characterization. Specific Aim 2: New instrumentation and methodologies for tandem mass spectrometry of peptides and proteins in "hybrid" tandem mass spectrometers. Specific Aim 3: Development of novel approaches for tandem mass spectrometry of proteins ionized via matrix-assisted laser desorption ionization (MALDI). These aims will be met by the development of novel ion/ion reactions for ion transformation, an expanded range of ion activation methods, instrument development to support the new ion chemistries, and the development of new approaches to protein identification/characterization that are enabled by the advances in ion chemistry and instrumentation. PUBLIC HEALTH RELEVANCE: This project is directed at improved protein identification/characterization. The ability to identify a protein and to characterize its post-translational modifications (i.e., identities and locations of modifications) underlie much of fundamental biomedical research. This capability is also key to the identification of protein biomarkers and their correlation with disease. This project seeks to address this task by greatly expanding the information content accessible via mass spectrometry.