Middle-down mass spectrometry (MD MS) has emerged as a promising alternative to classical bottom-up approaches for protein characterization. Middle-level experiments after enzymatic digestion are routinely used for subunit analysis of monoclonal antibody (mAb)-related compounds, providing information on drug load distribution and average drug-to-antibody ratio (DAR). However, peptide mapping is still the gold standard for primary amino acid sequence assessment, post-translational modifications (PTM), and drug conjugation identification and localization. However, peptide mapping strategies can be challenging when dealing with more complex and heterogeneous mAb formats, like antibody-drug conjugates (ADCs). We report here, for the first time, MD MS analysis of a third-generation site-specific DAR4 ADC using different fragmentation techniques, including higher-energy collisional- (HCD), electron-transfer (ETD) dissociation and 213 nm ultraviolet photodissociation (UVPD). UVPD used as a standalone technique for ADC subunit analysis afforded, within the same liquid chromatography-MS/MS run, enhanced performance in terms of primary sequence coverage compared to HCD- or ETD-based MD approaches, and generated substantially more MS/MS fragments containing either drug conjugation or glycosylation site information, leading to confident drug/glycosylation site identification. In addition, our results highlight the complementarity of ETD and UVPD for both primary sequence validation and drug conjugation/glycosylation site assessment. Altogether, our results highlight the potential of UVPD for ADC MD MS analysis for drug conjugation/glycosylation site assessment, and indicate that MD MS strategies can improve structural characterization of empowered next-generation mAb-based formats, especially for PTMs and drug conjugation sites validation.

中文翻译：

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用中向下质谱法鉴定位点特异性抗体-药物结合物的药物结合位点的案例研究。

中下质谱法（MD MS）已成为一种有前途的自下而上的蛋白质表征方法的替代方法。酶消化后的中级实验通常用于单克隆抗体（mAb）相关化合物的亚基分析，提供有关药物载量分布和平均药物与抗体比率（DAR）的信息。但是，肽图分析仍然是主要氨基酸序列评估，翻译后修饰（PTM）以及药物结合鉴定和定位的金标准。但是，在处理更复杂和异构的mAb格式（例如抗体-药物偶联物（ADC））时，肽图分析策略可能会具有挑战性。我们在这里首次报告使用不同的碎片技术对第三代特定于站点的DAR4 ADC的MD MS分析，包括高能碰撞（HCD），电子转移（ETD）解离和213 nm紫外线光解离（UVPD）。与基于HCD或ETD的MD方法相比，UVPD作为ADC亚基分析的独立技术，在相同的液相色谱-MS / MS运行中提供了增强的主要序列覆盖性能，并产生了更多的MS / MS片段包含药物共轭或糖基化位点信息，从而使您有把握地确定药物/糖基化位点。此外，我们的结果突出了ETD和UVPD在一级序列验证和药物结合/糖基化位点评估方面的互补性。总而言之，我们的结果突出了UVPD在ADC MD MS分析中用于药物结合/糖基化位点评估的潜力，