Proteome Center Rostock, University Medicine Rostock, Schillingallee 69, 18057, Rostock, Germany.
Institute of Immunology, University Medicine Rostock, Schillingallee 70, 18057, Rostock, Germany.
J Am Soc Mass Spectrom. 2017 Aug;28(8):1612-1622. doi: 10.1007/s13361-017-1654-7. Epub 2017 Jun 14.
Intact transition epitope mapping (ITEM) enables rapid and accurate determination of protein antigen-derived epitopes by either epitope extraction or epitope excision. Upon formation of the antigen peptide-containing immune complex in solution, the entire mixture is electrosprayed to translate all constituents as protonated ions into the gas phase. There, ions from antibody-peptide complexes are separated from unbound peptide ions according to their masses, charges, and shapes either by ion mobility drift or by quadrupole ion filtering. Subsequently, immune complexes are dissociated by collision induced fragmentation and the ion signals of the "complex-released peptides," which in effect are the epitope peptides, are recorded in the time-of-flight analyzer of the mass spectrometer. Mixing of an antibody solution with a solution in which antigens or antigen-derived peptides are dissolved is, together with antigen proteolysis, the only required in-solution handling step. Simplicity of sample handling and speed of analysis together with very low sample consumption makes ITEM faster and easier to perform than other experimental epitope mapping methods. Graphical Abstract ᅟ.
完整的表位作图（ITEM）通过表位提取或表位切除，能够快速、准确地确定蛋白质抗原衍生的表位。在溶液中形成含抗原肽的免疫复合物后，将整个混合物进行电喷雾，将所有成分都转化为质子化离子进入气相。在气相中，根据抗体-肽复合物的质量、电荷和形状，通过离子迁移漂移或四极离子过滤，将其与未结合的肽离子分离。随后，通过碰撞诱导碎裂使免疫复合物解离，并在质谱仪的飞行时间分析器中记录“复合物释放肽”（实际上就是表位肽）的离子信号。将抗体溶液与溶解抗原或抗原衍生肽的溶液混合，以及抗原蛋白水解，是唯一需要在溶液中进行的处理步骤。样品处理的简单性和分析的速度以及非常低的样品消耗，使得 ITEM 比其他实验性表位作图方法更快、更容易操作。
