Characterizing Viruses with Mass Spectrometry

我们提出的研究目标是开发新的病毒鉴定方法，最初我们将重点研究人鼻病毒(HRV)、鸡舍病毒(FHV)和腺病毒，并进一步将这项技术用作鉴定病毒和病毒细胞受体的通用方法。一种方法是使用抗体或核酸反应性系链将HRV病毒从溶液中选择性地提取到平板上，然后分析蛋白质分解的衣壳蛋白。从本质上讲，这将发展为一种从生物液中提取和鉴定病毒的两步分析方法(结合和质谱仪)。此外，通过将HRV和腺病毒连接到表面，我们计划进行与细胞蛋白提取物的亲和力实验，目的是通过蛋白质质量图谱识别细胞受体。我们的研究目标更具体的概述如下：1.开发附着在固体载体上的亲和力方法来选择性地捕获病毒。目标病毒系统将从溶液中提取，并通过其衣壳蛋白的蛋白水解质谱图进行鉴定。解吸/电离技术，如基质辅助激光解吸/电离(MALDI)和硅上解吸/电离(DIOS)将首先使用。这些分析也将使用液相色谱串联质谱仪，以便对不同种类的蛋白质样品进行分析。由于大多数病毒感染没有快速、廉价的筛查方法，这种方法将进一步发展为识别病毒的一般方法。2.用固定化病毒鉴定细胞蛋白受体。整个固定化腺病毒和HRV将受到细胞提取物的影响，随后去除未结合的物质，并随后分析与病毒结合的蛋白质。蛋白质分解之后的质量分析和数据库搜索将使病毒受体的特征得以确定。

The goal of our proposed research is to develop novel approaches for characterizing viruses, initially we will focus on human rhinovirus (HRV), flock house virus (FHV) and adenovirus, and further, use this technology as a general assay for the identification of viruses as well as viral cellular receptors. One approach is the selective extraction of HRV viruses from solution onto a plate using antibodies or nucleic acid reactive tethers followed by analysis of the proteolyzed capsid proteins. Essentially, this would be developed as a two-step assay (binding combined with mass spectrometry) to extract and identify viruses from biofluids. Furthermore, by tethering HRV and adenovirus to a surface we plan to perform affinity experiments with cellular protein extracts with the goal of identifying cellular receptors via protein mass mapping. A more specific outline of our research aims are as follows: 1. Develop affinity approaches attached to a solid support to selectively capture viruses. The target virus systems will be extracted from the solution and identified by proteolytic mass mapping of their capsid proteins. Desorption/ionization techniques such as matrix-assisted laser desorption/ionization (MALDI) and desorption/ionization on silicon (DIOS) will be used initially. Liquid chromatography tandem mass spectrometry will also be used for these analyses to allow for the analyses of heterogeneous protein samples. As there are no rapid, inexpensive screens for most viral infections this approach will be further developed as a general method for identifying viruses. 2. Identify cellular protein receptors using immobilized virus. Whole immobilized adenovirus and HRV will be subjected to cellular extracts, followed by removal of unbound material and subsequent analysis of the proteins bound to the virus. Proteolysis followed by mass analysis and database searching will allow for the characterization of viral receptors.