Hochleitner E O, Borchers C, Parker C, Bienstock R J, Tomer K B
National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Protein Sci. 2000 Mar;9(3):487-96. doi: 10.1110/ps.9.3.487.
A combination of epitope excision, epitope extraction, and differential chemical modification followed by mass spectrometric peptide mapping was used for the characterization of a discontinuous epitope that is recognized by the mouse anti-HIV-p24 monoclonal antibody 5E2.A3. In epitope excision, the protein is first bound to an immobilized antibody and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the antibody. After epitope excision of the p24-5E2.A3 complex with endoproteinase Lys-C, a large fragment remained affinity bound corresponding to amino acids 1-158 of HIV-p24 (fragment 1-158). Further digestion, however, resulted in loss of affinity. Moreover, no affinity-bound fragments were observed after an epitope extraction experiment. These data from the epitope excision and extraction experiments suggest that the epitope is discontinuous. For the further characterization of the epitope, amino groups in the epitope-containing fragment were acetylated in both the affinity bound and free states followed by mass spectrometric analysis. Two successive acetylation reactions were performed: (1) the first used a low molar excess of acetic anhydride, and (2) the second, after separation from the antibody, a high molar excess of its hexadeuteroderivative. This isotopic labeling procedure, in combination with high resolution mass spectrometry, allowed the precise determination of relative reactivities of amino groups. In this study, no differences were observed in the ranking of the relative reactivities of five lysine residues. However, the N-terminal amino group was found to be part of the discontinuous epitope.
将表位切除、表位提取和差异化学修饰相结合，随后进行质谱肽图分析，用于表征一种不连续表位，该表位可被小鼠抗HIV-p24单克隆抗体5E2.A3识别。在表位切除中，蛋白质首先与固定化抗体结合，然后用蛋白水解酶消化。在表位提取中，蛋白质首先被消化，随后与抗体反应。用内肽酶Lys-C对p24-5E2.A3复合物进行表位切除后，一个大的片段仍保持亲和结合，对应于HIV-p24的1-158位氨基酸（片段1-158）。然而，进一步消化导致亲和力丧失。此外，在表位提取实验后未观察到亲和结合的片段。这些来自表位切除和提取实验的数据表明该表位是不连续的。为了进一步表征该表位，对含表位片段中的氨基在亲和结合态和游离态均进行乙酰化，随后进行质谱分析。进行了两次连续的乙酰化反应：（1）第一次使用低摩尔过量的乙酸酐，（2）第二次，在与抗体分离后，使用高摩尔过量的其十六氘代衍生物。这种同位素标记方法与高分辨率质谱相结合，能够精确测定氨基的相对反应性。在本研究中，五个赖氨酸残基的相对反应性排名未观察到差异。然而，发现N端氨基是不连续表位的一部分。
