A general strategy for epitope mapping by direct MALDI-TOF mass spectrometry using secondary antibodies and cross-linking
A general strategy for epitope mapping by direct MALDI-TOF mass spectrometry using secondary antibodies and cross-linking
复制标题
DOI：
10.1021/ac010258n
复制
发表时间：
2001-08-15
影响因子：
7.4
通讯作者：
Tomer, KB
中科院分区：
文献类型：
--
作者：
Peter, JF;Tomer, KB
有限蛋白分解和MALDI-TOF质谱仪的结合已成为确定表位的重要工具，但与高纯度抗体配合使用效果最好。在这里，我们报告了使用捕获抗体来减少在表位的质谱学测定中对抗体纯化的需要。在这一新方法中，使用共价结合到琼脂糖珠上的二次Fc特异性抗体来捕获一次抗体(感兴趣的抗体)。捕获后，这两种抗体是交叉连接的。然后将抗原与固定的抗体结合，并使用几种连续的蛋白水解酶进行蛋白分解。在这项研究中，这一策略是用粗制的小鼠抗ACTH免疫球蛋白溶液和促肾上腺皮质激素(ACTH)来演示的。与以前抗体直接与活化的珠子结合的方法相比，新的方法(1)结合的抗体与ACM的结合能力更高，(2)不需要对目标抗体进行纯化，(3)显著减少了MALDI质谱图中的化学背景。
The combination of limited proteolysis and MALDI-TOF mass spectrometry has become an important tool for the determination of epitopes but works best with highly purified antibodies. Here we report the use of capture antibodies to reduce the need for purification of the antibody in the mass spectrometric determination of the epitope. In this new method, a secondary Fc-specific antibody, covalently bound to Sepharose beads, is used to capture the primary antibody (the antibody of interest). After capture, the two antibodies are cross-linked. The antigen is then bound to the immobilized antibodies and subjected to proteolysis using several successive proteinases. In this study, this strategy is demonstrated with a crude mouse anti-ACTH IgG solution and adrenocorticotropin (ACTH). Comparing this strategy with previous methods where the antibody is bound directly to activated beads, the new method (1) results in a higher binding capacity of the bound antibody to ACM, (2) does not require purification of the antibody of interest, and (3) dramatically reduces the chemical background in the MALDI mass spectra.