Rapid Membrane-Based Digestion and Purification For LC-MS Protein Analysis

Glycosylation plays a critical role in the bioactivity of proteins. For example, glycosylation of the SARS-CoV-2 Spike receptor-binding domain affects viral entry into host cells. Similarly, the glycosylation of erythropoietin and monoclonal antibodies significantly influences their pharmacokinetic and pharmacodynamic properties. The bottom-up approach, which enables the attachment of glycans to peptides, is one of the most straightforward methods for studying protein glycosylation by liquid chromatography-mass spectrometry (LC-MS). In addition to identifying non-glycosylated peptides, it facilitates the analysis of site-specific glycosylation. However, this approach typically requires several hours or even overnight digestion. The digestion process becomes even more prolonged when multiple proteases are needed, which significantly hampers the efficiency of protein glycosylation analysis.

The research presented in this dissertation utilized protease-immobilized membranes to enable rapid protein digestion. This method achieves comparable glycan identification and semi-quantitative assessments to conventional overnight in-solution digestion. Further studies explored the use of tandem membranes, consisting of trypsin-immobilized membranes and C18-derivatized membranes, to streamline protein digestion and peptide purification. During centrifugation, proteins are initially digested in the first layer (trypsin-immobilized membranes), and the resulting peptides are subsequently enriched in the second layer (C18-derivatized membranes). Most salts and contaminants pass through the membranes and are effectively removed from the protein digests. This technique has been successfully applied to the sequence and glycosylation analysis of commercial monoclonal antibodies (Kanjinti, Bevacizumab, and Rituximab), as well as to the analysis of host cell proteins and in-house expressed antibodies. This prototype could prove valuable for real-time monitoring of product glycosylation, ensuring the consistency and efficacy of therapeutic agents.

糖基化在蛋白质的生物活性中扮演着至关重要的角色。例如，SARS-CoV-2刺突蛋白受体结合域的糖基化会影响病毒进入宿主细胞。同样，促红细胞生成素和单克隆抗体的糖基化对其药代动力学和药效学特性具有显著影响。自下而上的方法，即通过液相色谱-质谱联用（LC-MS）研究蛋白质糖基化时将糖苷连接到肽上的方法，是研究蛋白质糖基化的最直接方法之一。除了识别非糖基化肽段外，该方法还促进了位点特异性糖基化的分析。然而，这种方法通常需要数小时甚至整夜进行消化处理。当需要多种蛋白酶时，消化过程变得更加漫长，这显著阻碍了蛋白质糖基化分析的效率。

本论文中的研究利用了蛋白酶固定化膜以实现蛋白质的快速消化。该方法在糖苷鉴定和半定量评估方面与传统的过夜溶液消化相当。进一步的研究探讨了串联膜的使用，该膜由胰蛋白酶固定化膜和C18衍生化膜组成，以简化蛋白质消化和肽段纯化。在离心过程中，蛋白质首先在第一层（胰蛋白酶固定化膜）中消化，随后产生的肽段在第二层（C18衍生化膜）中富集。大部分盐和杂质通过膜，并有效地从蛋白质消化物中去除。这项技术已成功应用于商用单克隆抗体（康吉宁、贝伐珠单抗和利妥昔单抗）的序列和糖基化分析，以及宿主细胞蛋白和室内表达抗体的分析。该原型对于产品糖基化的实时监测具有潜在价值，确保治疗剂的稳定性和有效性。