Sadeghi Seyed Amirhossein, Fang Fei, Tabatabaeian Nimavard Reyhane, Wang Qianyi, Zhu Guijie, Saei Amir Ata, Sun Liangliang, Mahmoudi Morteza
Department of Chemistry, Michigan State University, East Lansing, MI, USA.
Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.
Nat Protoc. 2025 Aug 5. doi: 10.1038/s41596-025-01229-6.
The protein corona is a layer of biomolecules-primarily proteins-that adsorbs to nanoparticle (NP) surfaces in biological fluids. If the purpose of the NP is therapeutic, this can have a profound effect on its biological activity and function in vivo. Protein corona formation can also be exploited for diagnostic purposes and to differentially enrich proteins for biomarker discovery. For all of these applications, it is useful to determine which proteins, and which specific proteoforms, bind to different types of NP. The traditional mass spectrometry (MS)-based bottom-up proteomics does not accurately identify specific proteoforms within the protein corona. This limitation impedes the nanomedicine field's ability to precisely predict the biological fate and pharmacokinetics of nanomedicines and their effectiveness in early-stage biomarker discovery and disease detection because many different proteoforms of the same gene could exist in the corona, and they have divergent biological functions. Here, we describe how to use capillary zone electrophoresis (CZE)-MS-based top-down proteomics to characterize the proteoform landscape of the protein corona. Our procedures detail the recovery of intact proteoforms from NP surfaces by using detergent-assisted proteoform elution and the measurement of these proteoforms by using CZE-tandem MS (MS/MS) and CZE-high-field asymmetric waveform ion mobility spectrometry (FAIMS)-MS/MS. The entire workflow is completed within 3-4 d. Using this protocol, hundreds of proteoforms from the protein corona of polystyrene NPs can be identified. Distinct protein corona proteoform profiles were observed from NPs with different physicochemical properties. The addition of FAIMS is beneficial for more in-depth proteoform characterization.
蛋白质冠层是一层生物分子——主要是蛋白质——它们吸附在生物流体中的纳米颗粒（NP）表面。如果NP的用途是治疗性的，这可能会对其在体内的生物活性和功能产生深远影响。蛋白质冠层的形成也可用于诊断目的，并用于差异富集蛋白质以发现生物标志物。对于所有这些应用，确定哪些蛋白质以及哪些特定的蛋白质变体与不同类型的NP结合是很有用的。传统的基于质谱（MS）的自下而上蛋白质组学不能准确识别蛋白质冠层内的特定蛋白质变体。这一局限性阻碍了纳米医学领域精确预测纳米药物的生物命运和药代动力学以及它们在早期生物标志物发现和疾病检测中的有效性的能力，因为同一基因的许多不同蛋白质变体可能存在于蛋白质冠层中，并且它们具有不同的生物学功能。在这里，我们描述了如何使用基于毛细管区带电泳（CZE）-MS的自上而下蛋白质组学来表征蛋白质冠层的蛋白质变体景观。我们的程序详细介绍了通过使用去污剂辅助的蛋白质变体洗脱从NP表面回收完整的蛋白质变体，以及通过使用CZE串联质谱（MS/MS）和CZE-高场不对称波形离子迁移谱（FAIMS）-MS/MS对这些蛋白质变体进行测量。整个工作流程在3-4天内完成。使用该方案，可以鉴定出聚苯乙烯NP蛋白质冠层中的数百种蛋白质变体。从具有不同物理化学性质的NP中观察到了不同的蛋白质冠层蛋白质变体谱。添加FAIMS有利于更深入的蛋白质变体表征。
