Peter J F, Tomer K B
National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, North Carolina 27709, USA.
Anal Chem. 2001 Aug 15;73(16):4012-9. doi: 10.1021/ac010258n.
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 ACTH, (2) does not require purification of the antibody of interest, and (3) dramatically reduces the chemical background in the MALDI mass spectra.
有限蛋白酶解与基质辅助激光解吸电离飞行时间质谱联用已成为确定表位的重要工具，但该方法在使用高度纯化的抗体时效果最佳。在此，我们报告了在表位的质谱测定中使用捕获抗体来减少抗体纯化需求的情况。在这种新方法中，一种与琼脂糖珠共价结合的二级Fc特异性抗体用于捕获一级抗体（目标抗体）。捕获后，两种抗体交联。然后将抗原与固定化抗体结合，并使用几种连续的蛋白酶进行蛋白酶解。在本研究中，用粗制的小鼠抗促肾上腺皮质激素IgG溶液和促肾上腺皮质激素（ACTH）证明了该策略。将该策略与之前抗体直接结合到活化珠上的方法进行比较，新方法（1）使结合抗体对ACTH的结合能力更高，（2）不需要纯化目标抗体，（3）显著降低了基质辅助激光解吸电离质谱中的化学背景。
