Jang Eunju, Ma Fengfei, Tomazela Daniela, Fayadat-Dilman Laurence, Al-Sayah Mohammad Ahmed
Protein Sciences, Discovery Biologics, Merck & Co., Inc., South San Francisco, California 94080, United States.
Discovery Analytical Research, Analytical Enabling Capabilities, Merck & Co., Inc., South San Francisco, California 94080, United States.
J Am Soc Mass Spectrom. 2024 Dec 4;35(12):3002-3009. doi: 10.1021/jasms.4c00303. Epub 2024 Sep 26.
Posttranslational modifications (PTMs) are potential critical quality attributes in biotherapeutic development, as they can affect drug efficacy and safety. Tyrosine sulfation plays a critical role in protein-protein interactions and has been found on many surface receptors as well as antibody complementarity-determining regions (CDR). However, the presence and function of tyrosine sulfation in therapeutic proteins have not been broadly investigated due to difficulties in detecting the modification. Here, we establish an integrated strategy to identify tyrosine sulfation in biotherapeutic proteins. In silico prediction was used to estimate possible modification sites, followed by the elucidation with intact LCMS and native SCX-MS. The combination of these three steps takes less than 1 h, which provides quick and confident preliminary detection of potential CQAs. Taking NB1 as an example, three +80 Da mass shifts were observed from intact mass analysis and three acidic peaks were monitored by SCX, allowing confirmation of modification as either phosphorylation or sulfation. Peptide mapping, Fe-IMAC enrichment, and dephosphorylation were further conducted to provide improved signal intensity and differentiation of modification such as sulfation or phosphorylation. With this integrated strategy, we were able to identify for the first time both tyrosine sulfation and serine phosphorylation in one therapeutic protein.
翻译后修饰（PTMs）在生物治疗药物开发中是潜在的关键质量属性，因为它们会影响药物的疗效和安全性。酪氨酸硫酸化在蛋白质-蛋白质相互作用中起关键作用，并且已在许多表面受体以及抗体互补决定区（CDR）中发现。然而，由于检测这种修饰存在困难，治疗性蛋白质中酪氨酸硫酸化的存在和功能尚未得到广泛研究。在此，我们建立了一种综合策略来鉴定生物治疗蛋白质中的酪氨酸硫酸化。利用计算机预测来估计可能的修饰位点，随后通过完整液相色谱-质谱联用（LCMS）和天然强阳离子交换-质谱（SCX-MS）进行阐明。这三个步骤相结合耗时不到1小时，可对潜在关键质量属性进行快速且可靠的初步检测。以NB1为例，通过完整质量分析观察到三个+80 Da的质量位移，通过SCX监测到三个酸性峰，从而确认修饰为磷酸化或硫酸化。进一步进行肽图分析、铁离子固定化金属亲和色谱（Fe-IMAC）富集和去磷酸化，以提高信号强度并区分硫酸化或磷酸化等修饰。通过这种综合策略，我们首次在一种治疗性蛋白质中鉴定出酪氨酸硫酸化和丝氨酸磷酸化。
