Characterizing Viruses with Mass Spectrometry

我们提出的研究的目标是开发表征病毒的新方法，最初我们将专注于人鼻病毒（HRV），鸡舍病毒（FHV）和腺病毒，并进一步使用该技术作为鉴定病毒以及病毒细胞受体的通用检测方法。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提取到板上，然后分析蛋白水解的衣壳蛋白。从本质上讲，这将被开发为两步测定法（结合质谱法），以从生物流体中提取和鉴定病毒。 此外，通过将HRV和腺病毒拴系到表面，我们计划用细胞蛋白提取物进行亲和实验，目的是通过蛋白质质量图谱鉴定细胞受体。 具体的研究目标如下：1.开发附着在固体支持物上的亲和方法，以选择性地捕获病毒。 将从溶液中提取靶病毒系统，并通过其衣壳蛋白的蛋白水解质量图谱鉴定。 最初将使用基质辅助激光解吸/电离（MALDI）和硅上解吸/电离（DIOS）等解吸/电离技术。 液相色谱串联质谱法也将用于这些分析，以允许分析异质蛋白样品。 由于大多数病毒感染没有快速，廉价的筛选方法，这种方法将进一步发展为识别病毒的通用方法。2.用固定化病毒鉴定细胞蛋白受体。将整个固定的腺病毒和HRV进行细胞提取，然后去除未结合的材料，并随后分析与病毒结合的蛋白质。蛋白水解，然后进行质量分析和数据库搜索，将允许病毒受体的表征。

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.