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（Glycosylation）在蛋白质的生物活性中发挥关键作用。例如，新冠病毒（SARS-CoV-2）刺突蛋白受体结合域的糖基化会影响病毒侵入宿主细胞的过程。类似地，促红细胞生成素（erythropoietin）与单克隆抗体（monoclonal antibodies）的糖基化，会显著影响其药代动力学（pharmacokinetic）与药效动力学（pharmacodynamic）特性。
自下而上法（bottom-up approach）可实现聚糖与肽段的结合，是目前通过液相色谱-质谱联用法（liquid chromatography-mass spectrometry, LC-MS）研究蛋白质糖基化的最简便方法之一。除可识别非糖基化肽段外，该方法还能实现位点特异性糖基化分析。不过该方法通常需要数小时甚至过夜的酶解过程；若需使用多种蛋白酶，酶解时长会进一步延长，极大阻碍了蛋白质糖基化分析的效率。
本论文所报道的研究采用固定化蛋白酶膜（protease-immobilized membranes）实现了蛋白质的快速酶解。该方法的聚糖识别能力与半定量评估效果，可与传统的过夜溶液内消化法相媲美。后续研究进一步探索了由固定化胰蛋白酶膜（trypsin-immobilized membranes）与C18衍生化膜（C18-derivatized membranes）组成的串联膜（tandem membranes）的应用，以简化蛋白质酶解与肽段纯化流程。在离心过程中，蛋白质首先在第一层膜（固定化胰蛋白酶膜）中完成酶解，所得肽段随后在第二层膜（C18衍生化膜）中得到富集。大部分盐类与杂质会透过膜层，从而从酶解产物中被有效去除。该技术已成功应用于商业单克隆抗体（Kanjinti、贝伐珠单抗（Bevacizumab）与利妥昔单抗（Rituximab））的序列与糖基化分析，同时也可用于宿主细胞蛋白（host cell proteins）与自研表达抗体的分析。该原型技术有望实现治疗性产品糖基化的实时监测，保障治疗药物的一致性与有效性。