Peptide therapeutics are a growing modality in the pharmaceutical industry and expanding these therapeutics to hit intracellular targets would require establishing cell permeability. Rapid measurement target-agnostic cell permeability of peptides is still analytically challenging. In this study, we demonstrate the development of a rapid high-throughput label-free methodology based on a MALDI-hydrogen-deuterium exchange mass spectrometry (MALDI-HDX-MS) approach to rank-order peptide cell membrane permeability using live THP-1 and AsPc-1 cells. Peptides were incubated in the presence of live cells and their permeability into the cells over time was measured by MALDI-HDX-MS. A differential hydrogen-deuterium exchange approach was used to distinguish the peptides outside of the cells from those inside. The peptides on the outside of the cells were labeled using sufficiently short exposure to deuterium oxide, while the peptides inside of the cells were protected from labeling as a result of permeation into the cells. The deuterium labeled and peak area ratios of unlabeled peptides were compared and plotted over time. The developed methodology, referred to as Cell-based Approach Membrane Permeability Assay (CAMPA), was applied to study an array of 24 diverse peptides including cell-penetrating peptides, stapled and macrocyclic peptides. The cell membrane permeability results observed by CAMPA were corroborated by previously reported in literature data. The CAMPA MALDI-MS analysis was fully automated including MS data processing using internally developed Python scripts. Moreover, CAMPA was demonstrated to be useful for differentiating passive and active cell transportation by using an endocytosis inhibitor in cell incubation media for selected peptides.

肽疗法在制药行业是一种不断发展的模式，将这些疗法扩展到细胞内靶点需要建立细胞通透性。肽的快速测量与靶标无关的细胞通透性在分析上仍然具有挑战性。在这项研究中，我们展示了一种基于 MALDI-氢-氘交换质谱 (MALDI-HDX-MS) 方法的快速高通量无标记方法的开发，该方法使用活 THP-1 对肽细胞膜通透性进行排序和 AsPc-1 细胞。在活细胞存在下孵育肽，并通过 MALDI-HDX-MS 测量它们随时间进入细胞的渗透性。使用差异氢-氘交换方法来区分细胞外的肽和细胞内的肽。细胞外部的肽使用足够短的氧化氘暴露进行标记，而细胞内部的肽由于渗透到细胞中而受到保护而不会被标记。比较未标记肽的氘标记和峰面积比并随时间作图。开发的方法，称为基于细胞的方法膜渗透性测定 (CAMPA)，用于研究 24 种不同肽的阵列，包括细胞穿透肽、钉合肽和大环肽。CAMPA 观察到的细胞膜通透性结果得到了先前报道的文献数据的证实。CAMPA MALDI-MS 分析是完全自动化的，包括使用内部开发的 Python 脚本进行 MS 数据处理。而且，