Department of Polymer Science and Engineering, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States.
Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States.
ACS Biomater Sci Eng. 2022 Jun 13;8(6):2489-2499. doi: 10.1021/acsbiomaterials.2c00125. Epub 2022 May 24.
The delivery of functional proteins to the intracellular space offers tremendous advantages for the development of new therapeutics but is limited by the passage of these large polar biomacromolecules through the cell membrane. Noncovalent polymer-protein binding that is driven by strong carrier-cargo interactions, including electrostatics and hydrophobicity, has previously been explored in the context of delivery of functional proteins. Appropriately designed polymer-based carriers can take advantage of the heterogeneous surface of protein cargoes, where multiple types of physical binding interactions with polymers can occur. Traditional methods of assessing polymer-protein binding, including dynamic light scattering, circular dichroism spectroscopy, and fluorescence-based assays, are useful in the study of new polymer-based carriers but face a number of limitations. We implement for the first time the method of covalent labeling-mass spectrometry (CL-MS) to probe intermolecular surface interactions within noncovalent polymer-protein complexes. We demonstrate the utility of CL-MS for establishing binding of an amphiphilic block copolymer to negatively charged and hydrophobic surface patches of a model protein, superfolder green fluorescent protein (sfGFP), using diethylpyrocarbonate as a pseudo-specific labeling reagent. In addition, we utilize this method to explore differences at the intermolecular surface as the ratio of polymer to protein increases, particularly in the context of defining effective protein delivery regimes. By promoting an understanding of the intermolecular interactions in polymer-protein binding and identifying sites where polymers bind to protein surfaces, noncovalent polymer carriers can be more effectively designed for protein delivery applications.
将功能性蛋白质递送到细胞内空间为开发新的治疗方法提供了巨大的优势，但受到这些大型极性生物大分子穿过细胞膜的限制。以前在功能性蛋白质递送上探索了基于非共价聚合物-蛋白质结合的方法，这种结合是由包括静电相互作用和疏水性在内的强载体-货物相互作用驱动的。适当设计的基于聚合物的载体可以利用蛋白质货物的异质表面，其中可以发生多种类型的与聚合物的物理结合相互作用。包括动态光散射、圆二色性光谱和基于荧光的测定在内的传统聚合物-蛋白质结合评估方法在新的基于聚合物的载体研究中很有用，但面临着许多限制。我们首次实施了共价标记-质谱（CL-MS）方法来探测非共价聚合物-蛋白质复合物中的分子间表面相互作用。我们使用二乙基焦碳酸酯作为伪特异性标记试剂，证明了 CL-MS 用于建立两亲嵌段共聚物与模型蛋白质超折叠绿色荧光蛋白（sfGFP）的带负电荷和疏水面斑之间的结合的有用性。此外，我们利用这种方法来探索随着聚合物与蛋白质的比例增加，分子间表面的差异，特别是在定义有效蛋白质递药方案的背景下。通过促进对聚合物-蛋白质结合中分子间相互作用的理解并确定聚合物与蛋白质表面结合的部位，可以更有效地设计非共价聚合物载体用于蛋白质递药应用。
