Fibrillation of human insulin B-chain by pulsed hydrogen-deuterium exchange mass spectrometry.
Fibrillation of human insulin B-chain by pulsed hydrogen-deuterium exchange mass spectrometry.
复制标题
通过脉冲氢-氘交换质谱法测定人胰岛素 B 链的颤动。
DOI：
10.1016/j.bpj.2022.10.042
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发表时间：
2022
影响因子：
3.4
通讯作者：
Topp,ElizabethM
中科院分区：
文献类型：
--
作者：
Renawala,HarshilK;Topp,ElizabethM
胰岛素在轻微不稳定的条件下形成淀粉样原纤维，B链残基被认为在胰岛素原纤维化中起重要作用。在此，使用脉冲氢-氘交换质谱法（HDX-MS）、远紫外圆二色谱法、硫磺素T（ThioT）荧光、浊度和可溶性部分测量来监测在强制降解条件（40°C，连续振荡）下人胰岛素B链（INSB）在酸性溶液（lmg/mL，pH 4.5）中的原纤化的动力学和机制。最初，INSB快速形成富含β-折叠的寡聚体，其被保护免于HD交换并显示弱的ThioT结合。随后的原纤维生长和成熟伴随着对HD交换的更大保护和更强的ThioT结合。通过消化酶消化氘代INSB，HDX-MS表明N-末端（1-11，1-15）和中心（12-15，16-25）片段早期参与原纤维形成相互作用，而C-末端片段（25-30）参与有限。结果提供了对应激条件下INSB原纤化过程中分子间相互作用和结构变化的机制理解，并证明了脉冲HDX-MS在探测肽原纤化方面的应用。
Insulin forms amyloid fibrils under slightly destabilizing conditions, and B-chain residues are thought to play an important role in insulin fibrillation. Here, pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS), far-UV circular dichroism spectroscopy, thioflavin T (ThioT) fluorescence, turbidity, and soluble fraction measurements were used to monitor the kinetics and mechanisms of fibrillation of human insulin B-chain (INSB) in acidic solution (1 mg/mL, pH 4.5) under stressed conditions (40°C, continuous shaking). Initially, INSB rapidly formedβ-sheet-rich oligomers that were protected from HD exchange and showed weak ThioT binding. Subsequent fibril growth and maturation was accompanied by even greater protection from HD exchange and stronger ThioT binding. With peptic digestion of deuterated INSB, HDX-MS suggested early involvement of the N-terminal (1–11, 1–15) and central (12–15, 16–25) fragments in fibril-forming interactions, whereas the C-terminal fragment (25–30) showed limited involvement. The results provide mechanistic understanding of the intermolecular interactions and structural changes during INSB fibrillation under stressed conditions and demonstrate the application of pulsed HDX-MS to probe peptide fibrillation.