Generating Informative Sequence Tags from Antigen-Binding Regions of Heavily Glycosylated IgA1 Antibodies by Native Top-Down Electron Capture Dissociation.
Generating Informative Sequence Tags from Antigen-Binding Regions of Heavily Glycosylated IgA1 Antibodies by Native Top-Down Electron Capture Dissociation.
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DOI：
10.1021/jasms.0c00461
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发表时间：
2021-06-02
影响因子：
3.2
通讯作者：
Heck AJR
中科院分区：
文献类型：
--
作者：
Greisch JF;den Boer MA;Beurskens F;Schuurman J;Tamara S;Bondt A;Heck AJR
免疫球蛋白A（伊加）包括一些最丰富的人类抗体，并在保护粘膜表面免受病原体侵害方面发挥重要作用。伊加亚类（称为IgA 1和IgA 2）重链的独特结构特征使它们能够通过连接J链进行连接，从而产生伊加二聚体以及更高的寡聚体。虽然分泌型sIgA寡聚体在乳汁和唾液中占主导地位，但IgA主要作为单体存在于血清中。目前没有方法允许解开潜在存在于人抗体库中的数百万个独特的IgA。获得其高变抗原结合区的明确序列读数是伊加鉴定的先决条件。我们在这里报告了一种质谱方法，使用电子捕获解离（ECD）产生直接读取序列梯的可变部分的轻链和重链的IgA 1，特别是功能关键的CDR 3区。我们直接比较了重度和不均匀N-和O-糖基化抗CD 20 IgA 1、相应N-糖基化抗CD 20 IgG 1及其Fab部分的天然自上而下ECD光谱。我们发现，虽然具有非常不同的MS 1光谱，所有四个物种的天然自上而下的ECD MS 2光谱几乎相同，裂解发生在重链和轻链的CDR 3和FR 4区域内。从完整的糖基化的IgA 1的序列信息的ECD数据，我们预见，本地自上而下的ECD将成为一个有价值的补充工具，从牛奶，唾液或血清的IgA 1从头测序。
Immunoglobulins A (IgA) include some of the most abundant human antibodies and play an important role in defending mucosal surfaces against pathogens. The unique structural features of the heavy chain of IgA subclasses (called IgA1 and IgA2) enable them to polymerize via the joining J-chain, resulting in IgA dimers but also higher oligomers. While secretory sIgA oligomers are dominant in milk and saliva, IgAs exist primarily as monomers in serum. No method currently allows disentangling the millions of unique IgAs potentially present in the human antibody repertoire. Obtaining unambiguous sequence reads of their hypervariable antigen-binding regions is a prerequisite for IgA identification. We here report a mass spectrometric method that uses electron capture dissociation (ECD) to produce straightforward-to-read sequence ladders of the variable parts of both the light and heavy chains of IgA1s, in particular, of the functionally critical CDR3 regions. We directly compare the native top-down ECD spectra of a heavily and heterogeneously N- and O-glycosylated anti-CD20 IgA1, the corresponding N-glycosylated anti-CD20 IgG1, and their Fab parts. We show that while featuring very different MS1 spectra, the native top-down ECD MS2 spectra of all four species are nearly identical, with cleavages occurring specifically within the CDR3 and FR4 regions of both the heavy and light chain. From the sequence-informative ECD data of an intact glycosylated IgA1, we foresee that native top-down ECD will become a valuable complementary tool for the de novo sequencing of IgA1s from milk, saliva, or serum.
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影响因子：
7.4
作者：
Shaw, Jared B.;Malhan, Neha;Voinov, Valery G.
通讯作者：
Voinov, Valery G.
影响因子：
14.9
作者：
Schymkowitz J;Borg J;Stricher F;Nys R;Rousseau F;Serrano L
通讯作者：
Serrano L
影响因子：
5.8
作者：
Klein, Joshua;Carvalho, Luis;Zaia, Joseph
通讯作者：
Zaia, Joseph
DOI：
10.1074/mcp.m114.039537
发表时间：
2014-11
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Bondt A;Rombouts Y;Selman MH;Hensbergen PJ;Reiding KR;Hazes JM;Dolhain RJ;Wuhrer M
通讯作者：
Wuhrer M
影响因子：
3.2
作者：
Pacholarz, Kamila J.;Peters, Shirley J.;Barran, Perdita E.
通讯作者：
Barran, Perdita E.