Parker Carol E, Tomer Kenneth B
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Mol Biotechnol. 2002 Jan;20(1):49-62. doi: 10.1385/MB:20:1:049.
Proteolytic digestion of proteins bound to immobilized antibodies, combined with matrix assisted laser desorption (MALDI) mass spectrometric identification of the affinity-bound peptides, can be a powerful technique for epitope determination. Binding of the protein to the antibody is done while the protein is in its native, folded state. A purified protein is not required for this procedure, because only proteins containing the antigenic determinant will bind to the antibody in the initial step. The method makes use of the resistance of the antibody to enzymatic digestion. Enzymatic cleavage products of the antigenic protein not containing the epitope are washed off the beads, leaving the epitope-containing fragments affinity bound to the immobilized antibody. Dissociation of the antigen-antibody complex prior to mass spectrometric analysis is unnecessary because the affinity-bound peptides are released by the MALDI matrix crystallization process, although the antibody remains covalently attached to the sepharose beads. This epitope-mapping protocol has been used in the determination of both continuous and discontinuous epitopes on both glycosylated and unglycosylated proteins.
对与固定化抗体结合的蛋白质进行蛋白酶消化，结合基质辅助激光解吸（MALDI）质谱法鉴定亲和结合的肽段，可成为一种用于确定表位的强大技术。蛋白质与抗体的结合是在蛋白质处于天然折叠状态时进行的。此过程不需要纯化的蛋白质，因为在初始步骤中只有含有抗原决定簇的蛋白质才会与抗体结合。该方法利用了抗体对酶消化的抗性。不含表位的抗原蛋白的酶切产物从珠子上被洗脱下来，留下含表位的片段与固定化抗体亲和结合。在质谱分析之前不需要解离抗原 - 抗体复合物，因为亲和结合的肽段通过MALDI基质结晶过程释放，尽管抗体仍共价连接在琼脂糖珠上。这种表位图谱分析方案已用于确定糖基化和非糖基化蛋白质上的连续和不连续表位。
