Complementary Methods for de Novo Monoclonal Antibody Sequencing to Achieve Complete Sequence Coverage.,Journal of Proteome Research

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Complementary Methods for de Novo Monoclonal Antibody Sequencing to Achieve Complete Sequence Coverage.
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2020-04-27 , DOI: 10.1021/acs.jproteome.0c00223
Jianhui Cheng ₁ , Lingyu Wang ₁ , Craig M Rive ₂ , Robert A Holt ₂ , Gregg B Morin ₂ , David D Y Chen ₁

Affiliation

Mass spectrometry is a powerful tool for de novo sequencing of novel proteins. Recent efforts in this area have mainly focused on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Here, we present an alternative method, capillary electrophoresis tandem mass spectrometry (CE-MS/MS), for sequencing novel monoclonal antibodies. Using less than 200 ng in total of tryptic digest sample in a triplicated measurement, CE-MS/MS with pH-mediated focusing successfully sequenced mAb infliximab with 100% sequence coverage and 100% accuracy for the light chain and 96% coverage and 93% accuracy for the heavy chain. It was also demonstrated that CE-MS/MS gives comparable results, and in some cases, even better results, as compared to LC-MS/MS when used as a standalone technique. A combined workflow using both CE-MS/MS and LC-MS/MS was also used to sequence a novel antibody, anti-CD-176, resulting in the first proposed sequence for this mAb.

中文翻译：

de Novo单克隆抗体测序的补充方法，可实现完整的序列覆盖。

质谱分析是从头测序新蛋白的强大工具。该领域的最新努力主要集中在液相色谱与串联质谱联用（LC-MS / MS）上。在这里，我们提出了一种替代方法，毛细管电泳串联质谱（CE-MS / MS），用于对新型单克隆抗体进行测序。通过三次测量总共使用不到200 ng的胰蛋白酶消化样品，带有pH介导聚焦的CE-MS / MS成功测序了单抗英夫利昔单抗，轻链覆盖率100％，准确度100％，覆盖率96％，覆盖率93％重链的准确性。还证明了与作为独立技术的LC-MS / MS相比，CE-MS / MS提供了可比的结果，在某些情况下甚至提供了更好的结果。

更新日期：2020-07-02

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