De Novo MS/MS Sequencing of Native Human Antibodies.
De Novo MS/MS Sequencing of Native Human Antibodies.
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人类本地抗体的从头开始的MS/MS测序。
DOI：
10.1021/acs.jproteome.6b00608
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发表时间：
2017-01-06
影响因子：
4.4
通讯作者：
Bandeira N
中科院分区：
文献类型：
--
作者：
Guthals A;Gan Y;Murray L;Chen Y;Stinson J;Nakamura G;Lill JR;Sandoval W;Bandeira N
发现治疗性人类单克隆抗体 (mAb) 的一种直接途径是在暴露于感染性试剂或疾病病因后，从幸存者/血清阳性个体中分离外周 B 细胞，然后进行单细胞测序或杂交瘤生成。然而，外周 B 细胞并不总是容易获得，并且仅占所有身体组织中总 B 细胞群的一小部分。尽管已经证明串联质谱 (MS/MS) 技术可以询问体内对抗原的完整多克隆抗体 (pAb) 反应，但当前所有方法都根据来自同一患者 B 细胞基因测序的数据库来识别 MS/MS 谱图。在这项概念验证研究中，我们证明了一种新型 MS/MS 抗体发现方法的可行性，该方法仅需要血清抗体，而不需要对遗传物质进行测序。通过糖蛋白 B 抗原亲和力纯化来自 CMV 暴露个体的外周 pAb，并根据 MS/MS 数据进行从头测序。然后在哺乳动物细胞中制造纯 MS 衍生的 mAb，通过抗原结合 ELISA 验证效力。有趣的是，我们发现这些 mAb 占供体 IgG 总量的 1-2%，但在对同一患者的记忆 B 细胞进行平行测序时未检测到。
One direct route for the discovery of therapeutic human monoclonal antibodies (mAbs) involves the isolation of peripheral B cells from survivors/sero-positive individuals after exposure to an infectious reagent or disease etiology followed by single-cell sequencing or hybridoma generation. Peripheral B cells, however, are not always easy to obtain and only represent a small percentage of the total B cell population across all bodily tissues. Although it has been demonstrated that tandem mass spectrometry (MS/MS) techniques can interrogate the full polyclonal antibody (pAb) response to an antigen in vivo, all current approaches identify MS/MS spectra against databases derived from genetic sequencing of B cells from the same patient. In this proof-of-concept study, we demonstrate the feasibility of a novel MS/MS antibody discovery approach in which only serum antibodies are required, without the need for sequencing of genetic material. Peripheral pAbs from a CMV exposed individual were purified by glycoprotein B antigen-affinity and de novo sequenced from MS/MS data. Purely MS-derived mAbs were then manufactured in mammalian cells to validate potency via antigen-binding ELISA. Interestingly, we found that these mAbs accounted for 1–2% of total donor IgG but were not detected in parallel sequencing of memory B cells from the same patient.
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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：
3.4
作者：
Dekker, Lennard;Wu, Si;Pasa-Tolic, Ljiljana
通讯作者：
Pasa-Tolic, Ljiljana
影响因子：
7
作者：
Guthals, Adrian;Clauser, Karl R.;Bandeira, Nuno
通讯作者：
Bandeira, Nuno
影响因子：
1.7
作者：
Datta, Ritendra;Bern, Marshall
通讯作者：
Bern, Marshall
影响因子：
7
作者：
Kim, Sangtae;Mischerikow, Nikolai;Pevzner, Pavel A.
通讯作者：
Pevzner, Pavel A.