Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet - Biomedicum, Solnavägen 9, 17165 Solna, Sweden.
Biological and Biomimetic Material Laboratory (BBML), Center for Sustainable Materials (SusMat), School of Materials Science and Engineering, Nanyang Technological University (NTU), Singapore, Singapore 637553.
Anal Chem. 2023 Jul 25;95(29):10869-10872. doi: 10.1021/acs.analchem.3c02384. Epub 2023 Jul 13.
Engineering liquid-liquid phase separation (LLPS) of proteins and peptides holds great promise for the development of therapeutic carriers with intracellular delivery capability but requires accurate determination of their assembly properties , usually with fluorescently labeled cargo. Here, we use mass spectrometry (MS) to investigate redox-sensitive coacervate microdroplets (the dense phase formed during LLPS) assembled from a short His- and Tyr-rich peptide. We can monitor the enrichment of a reduced peptide in dilute phase as the microdroplets dissolve triggered by their redox-sensitive side chain, thus providing a quantitative readout for disassembly. Furthermore, MS can detect the release of a short peptide from coacervates under reducing conditions. In summary, with MS, we can monitor the disassembly and cargo release of engineered coacervates used as therapeutic carriers without the need for additional labels.
工程化液-液相分离 (LLPS) 蛋白质和肽对于开发具有细胞内递药能力的治疗载体具有巨大的应用前景，但需要准确确定它们的组装特性，通常需要使用荧光标记的货物。在这里，我们使用质谱 (MS) 来研究由短的富含组氨酸和酪氨酸的肽组装而成的氧化还原敏感共凝聚微滴（LLPS 过程中形成的密集相）。我们可以监测到疏相中的还原肽的富集，因为微滴在其氧化还原敏感侧链的触发下溶解，从而为解组装提供定量读数。此外，MS 可以检测到在还原条件下短肽从共凝聚物中的释放。总之，通过 MS，我们可以监测作为治疗载体的工程共凝聚物的解组装和货物释放，而无需额外的标签。
