Papac D I, Hoyes J, Tomer K B
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Protein Sci. 1994 Sep;3(9):1485-92. doi: 10.1002/pro.5560030914.
We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.
我们开发了一种方法，通过对固定化抗原-抗体复合物进行原位蛋白酶解，然后结合基质辅助激光解吸电离飞行时间质谱（MALDI/TOF），快速鉴定抗体的抗原决定簇。将小鼠抗蛙皮素单克隆抗体固定在琼脂糖珠上，然后使抗原胃泌素释放肽（GRP）结合。展示了通过MALDI/TOF对固定化抗原-抗体复合物进行直接分析，并能够鉴定约1 pmol的结合GRP。为了鉴定表位，将固定化抗原-抗体复合物用胰蛋白酶、糜蛋白酶、嗜热菌蛋白酶和氨肽酶M进行蛋白酶解。蛋白酶解后，通过MALDI/TOF直接鉴定抗原中与抗体接触并受到蛋白酶解保护的部分。随后，用0.1 M甘氨酸缓冲液（pH 2.3）从固定化抗体上洗脱表位，通过反相高效液相色谱分离，并用MALDI/TOF确认其身份。使用这种方法，抗蛙皮素单克隆抗体的表位被证明由GRP的最后7-8个残基（HWAVGHLM-NH2）组成。
