Protein conjugates such as antibody-drugs conjugates (ADCs) represents the next generation of therapeutic proteins. They allow to combine the biological properties of the protein format with the characteristics of the conjugated ligands. The reaction implemented to couple ligands to the peptide backbone represents a crucial aspect of the production of protein conjugates, influencing the nature and the heterogeneity of the conjugates obtained. Here, we report the concomitant use of MALDI-TOF MS and LC-MS/MS analysis to investigate the chemical functionalization of human serum albumin (HSA) by the intermediate of lysine residues, previously used to generate biopharmaceutical agents for medical imaging. A kinetic was performed by collecting samples after different reaction times and analyzing them using the two techniques. MALDI-TOF MS analyses allowed estimating the number of conjugated ligands in a robust manner and assess the global functionalization kinetic on the intact protein level. Results demonstrated a maximum of 38 modified residues out of the 59 lysines available showing the limitation of the chemical functionalization. Consequently, LC-MS/MS analysis provided a site-specific characterization of the residues undergoing chemical modification. Data exhibited unique properties due to the presence of the ligands which allowed to identify without ambiguity the residues exhibiting different modification rate and enabled the identification of the unmodified lysine. Results were compared to the structure of HSA described from crystallography data. The comparison strongly suggested that accessibility is influencing the residues respective reactivity. The relevant complementarity of the different techniques could be emphasized in order to perform an extensive characterization concerning the evolution of the primary structure of the protein during the chemical reaction, providing an improved insight on the conjugation process and offering the potentiality to tune the reaction.

中文翻译：

---

使用补充质谱技术对治疗性白蛋白进行化学功能化的动力学和结构表征。

蛋白质结合物，例如抗体-药物结合物（ADC），代表了下一代治疗性蛋白质。它们允许将蛋白质形式的生物学特性与结合的配体的特征相结合。进行的使配体与肽主链偶联的反应代表了蛋白质缀合物生产的关键方面，影响了所得缀合物的性质和异质性。在这里，我们报告同时使用MALDI-TOF MS和LC-MS / MS分析来研究赖氨酸残基的中间体对人血清白蛋白（HSA）的化学功能化作用，该赖氨酸残基以前曾用于产生医学影像学的生物药剂。通过在不同的反应时间后收集样品并使用两种技术对其进行分析来进行动力学分析。MALDI-TOF MS分析可以以稳健的方式估算缀合配体的数量，并评估完整蛋白水平上的整体功能化动力学。结果表明，在可用的59种赖氨酸中，最多有38个修饰残基表明了化学功能化的局限性。因此，LC-MS / MS分析提供了经过化学修饰的残基的位点特异性表征。由于配体的存在，数据表现出独特的性质，该配体允许毫无歧义地鉴定出表现出不同修饰率的残基，并能够鉴定未修饰的赖氨酸。将结果与从晶体学数据描述的HSA的结构进行比较。该比较强烈表明可及性影响残基各自的反应性。