Direct Determination of Antibody Chain Pairing by Top-down and Middle-down Mass Spectrometry Using Electron Capture Dissociation and Ultraviolet Photodissociation.
Direct Determination of Antibody Chain Pairing by Top-down and Middle-down Mass Spectrometry Using Electron Capture Dissociation and Ultraviolet Photodissociation.
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DOI：
10.1021/acs.analchem.9b03129
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发表时间：
2020-01-07
影响因子：
7.4
通讯作者：
Voinov VG
中科院分区：
文献类型：
--
作者：
Shaw JB;Liu W;Vasil Ev YV;Bracken CC;Malhan N;Guthals A;Beckman JS;Voinov VG
与单克隆抗体（mAb）治疗剂的发现和开发相关的一个挑战是确定重链和轻链配对。MS仪器和MS/MS方法的进步极大地增强了分析大的完整蛋白质的能力，从而产生更详细和准确的蛋白质型表征。因此，完整抗体或F（ab′）2和Fab片段的直接询问具有显著简化治疗性mAb发现过程的潜力。在这里，我们首次证明了使用电子捕获解离（ECD）和157 nm紫外光解离（UVPD）有效切割连接mAb重链和轻链的二硫键的能力。完整mAb、Fab或F（ab′）2质量、完整LC和Fd质量以及CDR 3序列覆盖率的组合使得能够从单个实验和实验条件中确定重链和轻链配对。这些结果证明了自上而下和自下而上的蛋白质组学在显着简化治疗性抗体发现方面的潜力。
One challenge associated with the discovery and development of monoclonal antibody (mAb) therapeutics is the determination of heavy chain and light chain pairing. Advances in MS instrumentation and MS/MS methods have greatly enhanced capabilities for the analysis of large intact proteins yielding much more detailed and accurate proteoform characterization. Consequently, direct interrogation of intact antibodies or F(ab′)2 and Fab fragments has the potential to significantly streamline therapeutic mAb discovery processes. Here, we demonstrate for the first time the ability to efficiently cleave disulfide bonds linking heavy and light chains of mAbs using electron capture dissociation (ECD) and 157 nm ultraviolet photodissociation (UVPD). The combination of intact mAb, Fab, or F(ab′)2 mass, intact LC and Fd masses, and CDR3 sequence coverage enabled determination of heavy chain and light chain pairing from a single experiment and experimental condition. These results demonstrate the potential of top-down and middle-down proteomics to significantly streamline therapeutic antibody discovery.
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人类本地抗体的从头开始的MS/MS测序。
影响因子：
4.4
作者：
Guthals A;Gan Y;Murray L;Chen Y;Stinson J;Nakamura G;Lill JR;Sandoval W;Bandeira N
通讯作者：
Bandeira N
影响因子：
46.9
作者：
Cheung, Wan Cheung;Beausoleil, Sean A.;Polakiewicz, Roberto D.
通讯作者：
Polakiewicz, Roberto D.
影响因子：
7
作者：
Guthals, Adrian;Clauser, Karl R.;Bandeira, Nuno
通讯作者：
Bandeira, Nuno
影响因子：
1.8
作者：
Gunawardena, Harsha P.;Gorenstein, Lev;McLuckey, Scott A.
通讯作者：
McLuckey, Scott A.
影响因子：
1.8
作者：
Kalli, Anastasia;Hakansson, Kristina
通讯作者：
Hakansson, Kristina