Conformer selected Top Down Sequencing - A Novel Approach to Structural Proteomics

“软电离”技术的发展使质谱分析成为蛋白质组学研究的核心技术。电喷雾电离(ESI)-质谱在这个后基因组时代被广泛用于确定蛋白质的初级结构，并且已经真正成为所谓的“自下而上”蛋白质组学分析的基本工具。大量的努力和资源已经投入到基于质谱的蛋白质组学中，其中大部分依赖于所谓的“自下而上”表征，其中蛋白质被酶切成多肽进行质谱分析，然后通过数据库关联来识别（和量化）所研究的蛋白质。这种方法在分析上有很多优点；该方法具有高通量和高灵敏度，在许多研究中取得了成功，但也存在一些不足。也许最明显的是，“自下而上”的方法不能提供被分析蛋白质的活性折叠和相互作用的直接信息，这限制了从这些研究中获得的功能数据。质谱法的分析优势也适用于检测完整的蛋白质和复合物，并且有一个新兴的研究领域用这种方法鉴定蛋白质，即所谓的“自上而下”的方法，在质谱仪中对蛋白质进行测序。大多数自顶向下的方法在测序之前破坏蛋白质的功能形式，以实现更高的通量，并促进对高电荷母离子的更有效分析。这不是我们将要采取的路线，而是我们将探测完整的蛋白质和复合物，小心地使用纳米电喷雾电离（nESI）来保留溶液结构。该提案将发展所谓的“自上而下”的方法来检查蛋白质和蛋白质复合物的构象和动力学。该研究计划将加强主要基于溶液相的技术范围，以评估蛋白质结构和相互作用。方法学将评估蛋白质在溶液和无溶剂环境中的构象稳定性和动力学。需要资金和研究时间来构建用于测量构象的新型仪器，以及在从微秒到分钟的时间尺度上展开和折叠动力学。所提出的仪器也将能够光解质量和构象选择离子，并检测产物离子。技术发展之后的工作方案将侧重于两个领域：自顶向下结构蛋白质组学；结合光解离和离子迁移质谱（IM-MS）来绘制蛋白质结构和相互作用。2. 采用FRET结合IM-MS，以确定蛋白质展开途径和荧光标记物对蛋白质结构的影响。该设备将包括一个新型的双离子引导迁移率质谱仪，其中IM-MS将用于确定碰撞截面，光学方法将通过FRET和/或光解离来询问结构和稳定性。这种结合的IM-MS光激活方法将被称为photo-IM-MS。离子将通过ESI在外部产生，并转移到定制的离子迁移率质谱仪中。一旦进入IM-MS的双重漂移区，离子的漂移时间（在弱电场的影响下）与它们与缓冲气体的旋转平均碰撞截面有关。这种迁移率测量可以进行，或者离子将被“步进”到一个平行堆叠的环形离子引导漂移区（2SRIG），该区域有激光束穿过它以及光学检测。该区域的离子将与光相互作用，要么被光学检测到，要么被推回第一迁移细胞。在离开细胞离子将被转移到飞行时间质谱仪，因此将被检测为质量，电荷状态和横截面的函数，以及潜在的光学相互作用

The development of 'soft ionisation' techniques have positioned mass spectrometry as the central go-to technique for proteomic investigations. Electrospray ionisation (ESI)-MS is used extensively in this post-genomic era to determine the primary structure of proteins and has really become the essential tool in so called 'bottom-up' proteomic analysis. Extensive effort and resource has gone into mass spectrometry based proteomics, the majority of which relies on so called 'bottom-up' characterization where proteins are enzymatically cleaved into peptides for MS analysis followed by database correlation to identify (and quantify) the proteins under study. This approach has many analytical advantages; critically it is high throughput and sensitive and clearly has succeeded in many studies however it has some drawbacks. Perhaps the most obvious is that "bottom up" approaches cannot provide direct information on the active fold and interactions of the proteins being analysed, this limits the functional data that could be obtained from these studies. The analytical advantages of mass spectrometry also apply to its use to examine intact proteins and complexes and there is an emerging research field that identifies proteins this way - so called "top-down" methodologies where proteins are sequenced in the mass spectrometer. Most top down approaches destroy the functional form of the protein prior to sequencing, to enable higher throughput and to facilitate more productive analysis from higher charged parent ions. This is not quite the route we will take, rather we will probe intact proteins and complexes , with careful use of nano-electrospray ionisation (nESI) to retain solution structures. This proposal will develop so called "top down" methods to examine conformations and dynamics of proteins and protein complexes. The research program will enhance the gamut of predominantly solution-phase based techniques which evaluate protein structure and interactions. Methodologies will assess conformational stability, and dynamics of proteins both in solution and in a solvent free environment. Funds and research time are requested to construct novel instrumentation with which to measure conformations, and unfolding and refolding dynamics over timescales ranging from microseconds to minutes. The proposed instrument will also be able to photo-dissociate mass and conformer selected ions, and detect the product ions. The program of work following technology development will focus on two areas: 1. Top down structural proteomics; combining photo-dissociation and ion mobility mass spectrometry (IM-MS) to map protein structure and interactions. 2. Performing FRET combined with IM-MS, to determine protein unfolding pathways and the effect of fluorescent makers on protein structure.The equipment will comprise a novel duel ion guide mobility mass spectrometer, wherein IM-MS will be used to determine collision cross sections, and optical methods will interrogate structure and stability via FRET and/or photo dissociation. This combined IM-MS photo activation approach will be termed photo-IM-MS. Ions will be externally generated via ESI and transferred into a customised ion mobility mass spectrometer. Once in the duel drift region of the IM-MS, the drift time of ions (under the influence of a weak electric field) is related to their rotationally averaged collision cross section with the buffer gas. This mobility measurement can be made, or alternatively the ions will be 'stepped' into a parallel stacked ring ion guide drift region (2SRIG), which has a laser beam passing through it as well as optical detection. Ions in this region will interact with light and either be optically detected, or be pushed back into the first mobility cell. On exiting the cell ions will be transferred to a time-of-flight mass spectrometer and thus will be detected as a function of both mass, charge state, and cross section and potentially following optical interaction