Department of Chemistry & Environmental Science, New Jersey Institute of Technology, Newark, New Jersey 07102, United States.
Janssen Research & Development, The Janssen Pharmaceutical Companies of Johnson & Johnson, Spring House, Pennsylvania 19477, United States.
Anal Chem. 2021 Mar 2;93(8):3997-4005. doi: 10.1021/acs.analchem.0c04974. Epub 2021 Feb 16.
Recently, microdroplet reactions have aroused much interest because the microdroplet provides a unique medium where organic reactions could be accelerated by a factor of 10 or more. However, microdroplet reactions of proteins have been rarely studied. We report the occurrence of multiple-step reactions of a large protein, specifically, the digestion, reduction, and deglycosylation of an intact antibody, which can take place in microseconds with high reaction yields in aqueous microdroplets at room temperature. As a result, fast structural characterization of a monoclonal antibody, essential for assessing its quality as a therapeutic drug, can be enabled. We found that the IgG1 antibody can be digested completely by the IdeS protease in aqueous microdroplets in 250 microseconds, a 7.5 million-fold improvement in speed in comparison to traditional digestion in bulk solution (>30 min). Strikingly, inclusion of the reductant tris(2-carboxyethyl)phosphine in the spray solution caused simultaneous antibody digestion and disulfide bond reduction. Digested and reduced antibody fragments were either collected or analyzed online by mass spectrometry. Further addition of PNGase F glycosylase into the spray solution led to antibody deglycosylation, thereby producing reduced and deglycosylated fragments of analytical importance. In addition, glycated fragments of IgG1 derived from glucose modification were identified rapidly with this ultrafast digestion/reduction technique. We suggest that microdroplets can serve as powerful microreactors for both exploring large-molecule reactions and speeding their structural analyses.
最近，微滴反应引起了广泛的关注，因为微滴提供了一种独特的介质，可以将有机反应加速 10 倍甚至更多。然而，蛋白质的微滴反应却很少被研究。我们报告了一种大蛋白质的多步反应的发生，具体来说，就是完整抗体的消化、还原和去糖基化，这些反应可以在室温下的水性微滴中在微秒内以高反应收率发生。因此，可以实现对单克隆抗体的快速结构表征，这对于评估其作为治疗药物的质量至关重要。我们发现，在水性微滴中，IgG1 抗体可以在 250 微秒内被 IdeS 蛋白酶完全消化，与传统的在大量溶液中的消化相比，速度提高了 750 万倍（>30 分钟）。引人注目的是，在喷雾溶液中加入还原剂三(2-羧乙基)膦（tris(2-carboxyethyl)phosphine）会导致抗体的同时消化和二硫键还原。消化和还原的抗体片段可以在线收集或通过质谱进行分析。进一步将 PNGase F 糖苷酶添加到喷雾溶液中会导致抗体去糖基化，从而产生具有分析重要性的还原和去糖基化片段。此外，用这种超快消化/还原技术可以快速鉴定 IgG1 衍生的葡萄糖修饰的糖化片段。我们认为，微滴可以作为探索大分子反应和加速其结构分析的强大微反应器。
