Dhungana Suraj, Williams Jason G, Fessler Michael B, Tomer Kenneth B
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, DHHS, 111 T.W. Alexander Drive, PO Box 12233, Research Triangle Park, NC 27709, USA.
Methods Mol Biol. 2009;524:87-101. doi: 10.1007/978-1-59745-450-6_7.
The ability to accurately characterize an epitope on an antigen is essential to understand the pathogenesis of an infectious material, and for the design and development of drugs and vaccines. Emergence of a new contagious microbial or viral variant necessitates the need for robust identification and characterization of the antigenic determinant. Recent advances have made mass spectrometry (MS) a robust and sensitive analytical tool with high mass accuracy. The use of MS to characterize peptides and proteins has gained popularity in the research arena involving protein-protein interactions. Combining the modern mass spectrometric principles of protein-protein interaction studies with the classical use of limited proteolysis, a linear epitope on a peptide or a protein antigen can be accurately mapped in a short time, compared with other traditional techniques available for epitope mapping. Additionally, complete MS analyses can be achieved with very little sample consumption. Here we discuss the overall approach to characterize the detailed interaction between a linear antigen (either a peptide or a protein antigen) and its corresponding monoclonal antibody by using MS. The steps involved in epitope excision, epitope extraction, and indirect immunosorption are outlined thoroughly. Conditions required for MS analysis using either matrix assisted laser desorption ionization (MALDI) or electrospray ionization (ESI) sources are summarized, with special emphasis on the experimental protocols.
准确表征抗原上的表位对于理解传染性物质的发病机制以及药物和疫苗的设计与开发至关重要。新的传染性微生物或病毒变体的出现使得对抗抗原决定簇进行可靠的鉴定和表征成为必要。最近的进展使质谱（MS）成为一种具有高质量准确性的强大而灵敏的分析工具。在涉及蛋白质-蛋白质相互作用的研究领域，使用MS表征肽和蛋白质已变得越来越普遍。将蛋白质-蛋白质相互作用研究的现代质谱原理与有限蛋白酶解的经典应用相结合，与其他用于表位作图的传统技术相比，可以在短时间内准确地绘制肽或蛋白质抗原上的线性表位。此外，只需消耗极少的样品就能完成完整的MS分析。在此，我们讨论使用MS表征线性抗原（肽或蛋白质抗原）与其相应单克隆抗体之间详细相互作用的总体方法。详细概述了表位切除、表位提取和间接免疫吸附所涉及的步骤。总结了使用基质辅助激光解吸电离（MALDI）或电喷雾电离（ESI）源进行MS分析所需的条件，并特别强调了实验方案。
