Global Research Technologies, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark.
Department of Health Science and Technology, Aalborg University, Fredrik Bajers Vej 7, 9220 Aalborg, Denmark.
J Am Soc Mass Spectrom. 2021 Aug 4;32(8):1910-1918. doi: 10.1021/jasms.0c00257. Epub 2020 Oct 21.
High-molecular weight products (HMWP) are an important critical quality attribute in research and development of insulin biopharmaceuticals. We here demonstrate on two case studies of covalent insulin dimers, induced by Fe incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites. On the MS level, electron-transfer/higher-energy collision dissociation (EThcD) efficiently cleaved the interchain disulfide bonds in the dimers to reveal cross-link connectivities between chains. The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links. Identification of cross-link sites between chains at residue level was achievable for both dimers with MS analysis of MS fragments cleaved at the cross-link or additionally the interchain disulfide bonds. UVPD provided identification of cross-link sites in the Fe-induced dimer without MS, while cross-link site identification with MS was not possible for the UV light-induced dimer. Thus, using varied multistage approaches, it was discovered that in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain. In the Fe-induced dimer, Phe1 from both B-chains were cross-linked through a -CH-. The UV chromophoric side chain of Phe1 was indicated in the cross-link, explaining why UVPD-MS was effective in fragmenting the cross-link and nearby backbone bonds. Our results demonstrated that higher-energy collisional dissociation (HCD), EThcD, and UVPD combined with MS were powerful tools for direct de novo characterization of cross-linked insulin dimers.
高分子量产物（HMWP）是胰岛素生物制药研发中的一个重要关键质量属性。我们在这里通过两个案例研究证实了共价胰岛素二聚体，这些二聚体由铁孵育或紫外线（UV）光应激诱导，从头鉴定的顶部向下质谱（MS）工作流程可以识别交联类型和位置。在 MS 水平上，电子转移/更高能量碰撞解离（EThcD）有效地切割二聚体中的链间二硫键，以揭示链间交联的连接性。EThcD 和 213nm 紫外线光解离（UVPD）的联合利用有助于鉴定交联的化学组成。通过对在交联处或另外在链间二硫键处切割的 MS 片段进行 MS 分析，可以实现两个二聚体的链间残基交联位点的鉴定。UVPD 提供了无 MS 情况下 Fe 诱导二聚体中交联位点的鉴定，而 MS 对 UV 光诱导二聚体中交联位点的鉴定则不可行。因此，使用多种多步方法，发现 UV 光诱导二聚体中，A 链的 Tyr14 参与了-O-S-交联，其中硫来自 B 链的 Cys7 或 Cys19。在 Fe 诱导的二聚体中，两个 B 链的 Phe1 通过-CH-交联。Phe1 的 UV 发色侧链在交联中被指示，这解释了为什么 UVPD-MS 有效地切割交联和附近的骨架键。我们的结果表明，更高能量的碰撞解离（HCD）、EThcD 和 UVPD 与 MS 相结合是直接从头鉴定交联胰岛素二聚体的有力工具。
