STRUCTURE ELUCIDATION OF PROTEINS BY MASS SPECTROMETRY

描述：了解蛋白质在分子水平上的功能 在这一层面上，必须有切实可行的手段来探索其 结构和动力学特征。 肽酰胺 当蛋白质被置于D（2）O中时， 已被广泛用作蛋白质结构和动力学的探针。 目前通过该赠款资助的研究表明，酰胺 氢交换率可以通过质量以高精度确定 光谱法，这种方法提供了几个优势， 目前用于定量蛋白质中氢交换的其他方法。 例如，酰胺氢交换速率可以通过以下方式确定： 几乎任何大小的蛋白质，通常只需要皮摩尔 进行分析。 氢交换发生在从毫秒到数月的时间尺度上 可通过在以下文献中开发的蛋白质片段化/MS方法进行定量： 目前的方案。 经过一段时间的氘交换， 将pH降低至2-3以淬灭氢交换反应。 胃蛋白 将蛋白质片段化为短肽， 通过直接耦合HPLC电喷雾电离测定重量 质谱分析法来 测定肽的氘含量 从它们的分子量。 拟议调查的长期目标是进一步发展 蛋白质片段化/MS方法，使其成为主流工具 用于研究蛋白质结构和动力学。 实现这一 目标，蛋白质片段化/MS方法将适用于特定的 酰胺氢交换已经 证明是有用的 提出了四种不同类型的应用 为了证明可以使用蛋白质片段化/MS方法， 有利地研究蛋白质结构和动力学。 这些 应用包括[1]热力学和动力学的研究， 蛋白质解折叠，[2]一项研究膜结合蛋白质的设计， 检测蛋白质结合时发生的构象变化， 膜，[3]旨在识别蛋白质结构的研究 折叠的中间体， 蛋白质折叠，[4]一项旨在表征结构的研究 发生在非常大的蛋白质中的变化（M（r）124，000）。 此外，本发明还提供了一种方法， 已经提出了几个项目来提高灵敏度、速度 和蛋白质片段化/MS方法的空间分辨率。 是 重要的是要注意现有的测量酰胺氢的方法 蛋白质的交换率不适合任何提议的 应用.

DESCRIPTION: To understand the functions of proteins at the molecular level, it is necessary to have practical means for probing their structural and dynamical features. The rates at which peptide amide hydrogens undergo isotopic exchange when proteins are placed in D(2)O has been used extensively as a probe of protein structure and dynamics. Research currently funded through this grant has demonstrated that amide hydrogen exchange rates can be determined with high accuracy by mass spectrometry, and that this approach offers several advantages over other methods currently used to quantify hydrogen exchange in proteins. For example, amide hydrogen exchange rates can be determined for proteins of virtually any size, and typically requires only picomoles of protein for an analysis. Hydrogen exchange occurring on the time scale from milliseconds to months can be quantified by the protein fragmentation/MS method developed in the current program. Following a period of deuterium exchange in, the pH is decreased to 2-3 to quench the hydrogen exchange reaction. Pepsin is added to fragment the protein into short peptides whose molecular weights are determined by directly-coupled HPLC electrospray ionization mass spectrometry. The deuterium contents of the peptides are determined from their molecular weights. The longterm goal of the proposed investigation is to further develop the protein fragmentation/MS method so that it will become a mainstream tool for investigations of protein structure and dynamics. To achieve this goal, the protein fragmentation/MS method will be adapted to specific types of applications for which amide hydrogen exchange has already proved useful. Four different types of applications have been proposed to demonstrate that the protein fragmentation/MS approach can be used advantageously to investigate protein structure and dynamics. These applications include [1] a study of the thermodynamics and kinetics of protein unfolding, [2] a study of membrane-bound proteins designed to detect conformational changes occurring while the protein is bound to the membrane, [3] a study designed to identify structures of protein folding intermediates which will illuminate mechanisms through which proteins fold, and [4] a study designed to characterize structural changes occurring in a very large protein (M(r)124,000). In addition, several projects have been proposed to improve the sensitivity, speed and spatial resolution of the protein fragmentation/MS method. It is important to note that existing methods for measuring amide hydrogen exchange rates in proteins are not suited for any of the proposed applications.