PO.CH03.01 · 化学
使用微流控调制光谱法（MMS）优化 pH 低插入肽 pH 依赖性构象变化的检测
Optimizing the detection of pH-dependent conformational changes in pH-low insertion peptides using microfluidic modulation spectroscopy (MMS)
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摘要 Abstract
中文摘要
引言：胰腺导管腺癌（PDAC）的酸性微环境可被 pH 响应性肽靶向，这些肽在其带负电荷残基质子化时发生构象变化。虽然微流控调制光谱法（MMS）已成功用于检测大蛋白的高阶结构，但在可靠检测小肽（<30 个氨基酸）的二级结构方面仍有优化空间。我们利用两种 pH 响应性肽 V7 和 V3 以及一种无活性肽 K7，研究缓冲液条件和脂质体的存在对 MMS 检测这些小肽 pH 依赖性构象变化能力的影响。
方法：使用 CEM LibertyBlue 微波技术合成肽。制备 pH 7.4 和 6.6 的 10 mM 和 20 mM 磷酸盐缓冲液（PBS）。使用薄膜水合技术制备脂质体，然后用动态光散射（DLS）进行表征，测定其粒径、多分散指数和表面电位。使用 RedShiftBio Aurora MMS 系统在 pH 7.4 或 6.6 的 PBS 中，有或无脂质体的条件下分析 V7、V3 和 K7 的二级结构。每种缓冲液溶液配制含 1 mg/mL 肽，并在 MMS 读数前超声处理 30 分钟。使用 RedShiftBio 高阶结构和高斯分析软件比较每种肽在各种缓冲液条件下 pH 7.4 至 6.6 之间的构象变化。
结果：DLS 显示脂质体粒径为 122 nm，多分散指数为 0.207，表面电位为 +32.9 mV。在无脂质体存在的溶液中，无法充分检测 V7 和 V3 基于 pH 的预期构象变化。含 10⁶ 个脂质体/mL 浓度的 10 mM PBS 是表征肽的最佳溶液，其中 V7 和 V3 在 pH 6.6 时相比 7.4 时 α 折叠增加，而 K7 在 pH 6.6 时相比 7.4 时无变化或 α 折叠减少。
结论：含脂质体的低盐浓度溶液是肽 V7 和 V3 发生可被 MMS 检测的 pH 依赖性构象变化的理想环境。MMS 是一种相对快速的测定肽二级结构的方法，具有较低的定量限。
资助：R01CA2810190
查看英文原文 English abstract
Introduction: The acidic microenvironment of pancreatic ductal adenocarcinoma (PDAC) can be targeted by pH-responsive peptides that undergo conformational changes upon protonation of their negatively charged residues. While microfluidic modulation spectroscopy (MMS) has been successfully used to detect higher-order structure in large proteins, there is room for optimization in the reliable detection of secondary structure for small peptides (< 30 amino acids). We utilized two pH-responsive peptides, V7 and V3, and an inactive peptide, K7, to investigate the effects of buffer conditions and the presence of liposomes on the ability of MMS to detect pH-based conformational changes in these small peptides.
Methods: Peptides were synthesized using the CEM LibertyBlue microwave technology. 10 mM and 20 mM phosphate buffer solutions (PBS) were prepared at pH 7.4 and 6.6. Liposomes were prepared using the thin film hydration technique, and then characterized with dynamic light scattering (DLS) to find size, polydispersity index, and surface potential. The RedShiftBio Aurora MMS system was used to analyze the secondary structure of V7, V3, and K7 at either pH 7.4 or 6.6 PBS with liposomes or without liposomes. Each buffer solution was prepared with 1 mg/mL of peptide and sonicated for 30 minutes before MMS reading. RedShiftBio higher-order structure and Gaussian analysis software were used to compare conformational changes between pH 7.4 to 6.6 for each peptide in the various buffer conditions.
Results: DLS revealed the liposomes to be 122 nm with a polydispersity index of 0.207 and a surface potential of +32.9 mV. The expected conformational changes for V7 and V3 based on pH could not be adequately detected without the presence of liposomes in the solution. 10 mM PBS with a concentration of 10 6 liposomes per mL was the optimal solution for characterizing the peptides, with V7 and V3 experiencing an increase in alpha-fold at pH 6.6 compared to 7.4, and K7 experiencing either no change or less alpha-fold at pH 6.6 compared to 7.4.
Conclusion: A low salt concentration solution containing liposomes was the ideal environment for the peptides V7 and V3 to undergo a pH-dependent conformational change that could be detected by MMS. MMS is a relatively quick method for determining peptide secondary structure with a low limit of quantification.
Funding: R01CA2810190
利益披露 Disclosure
E. Sanderson, None..
R. Bynum, None..
H. Agarwal, None..
L. McNally, None.