Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays
Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays
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DOI：
10.1074/mcp.m113.033308
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发表时间：
2014-06-01
影响因子：
7
通讯作者：
Uhlen, Mathias
中科院分区：
文献类型：
--
作者：
Forsstrom, Bjorn;Axnas, Barbara Bislawska;Uhlen, Mathias
抗体对于蛋白质组学领域是重要的，既作为用于成像细胞、组织和器官的试剂，又作为用于基于质谱的技术中的亲和富集的捕获剂。重要的是获得关于抗体的结合位点（表位）和与非靶蛋白的潜在交叉反应性的基本见解。关于抗体的线性表位的知识也可用于例如开发涉及在质谱分析之前捕获从样品的胰蛋白酶裂解获得的肽的测定。在这里，我们描述了，第一次，肽阵列的设计和使用，涵盖所有的人类蛋白质的抗体特异性分析，基于并行的原位双稳态合成的共210万重叠肽。这允许分析单克隆和多克隆抗体的靶向和脱靶结合，并基于全氨基酸取代扫描进行表位的精确定位。分析表明，线性表位相对较短，仅限于5至7个残基，导致与对应于大量不相关人类蛋白质的肽的明显脱靶结合。然而，随后使用重组蛋白的分析表明，这些线性表位具有严格的构象成分，从而为我们提供了关于抗体如何结合其抗原的新见解。
Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on-and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.