Abstract：
In antibody-based drug research, a complete characterization of antibody proteoforms covering both the amino acid sequence and all posttranslational modifications remains a major concern. The usual mass spectrometry-based approach to achieve this goal is bottom-up proteomics, which relies on the digestion of antibodies but does not allow the diversity of proteoforms to be assessed. Middle-down and top-down approaches have recently emerged as attractive alternatives but are not yet mastered and thus used in routine by many analytical chemistry laboratories. The work described here aims at providing guidelines to achieve the best sequence coverage for the fragmentation of intact light and heavy chains generated from a simple reduction of intact antibodies using Orbitrap mass spectrometry. Three parameters were found crucial to this aim: the use of an electron-based activation technique, the multiplex selection of precursor ions of different charge states, and the combination of replicates.
摘要：
在基于抗体的药物研究中，涵盖氨基酸序列和所有翻译后修饰的抗体蛋白质形式的完整表征仍然是一个主要问题。实现这一目标的通常基于质谱的方法是自下而上的蛋白质组学，这依赖于抗体的消化，但不允许评估蛋白质形式的多样性。中下和自上而下的方法最近已成为有吸引力的替代方法，但尚未掌握，因此许多分析化学实验室常规使用。这里描述的工作旨在提供指南，以实现使用Orbitrap质谱法简单还原完整抗体产生的完整轻链和重链片段化的最佳序列覆盖。发现三个参数对此至关重要：使用基于电子的激活技术，不同电荷状态的前体离子的多路选择，和重复的组合。
