Kiselar J G, Downard K M
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461-1602, USA.
Anal Chem. 1999 May 1;71(9):1792-801. doi: 10.1021/ac9811120.
A rapid new approach is described that combines the selectivity and sensitivity of immunoaffinity and mass spectrometric based techniques for mapping protein epitopes. The approach alleviates the need to immobilize antibody, extract the antibody-peptide complex, and dissociate bound peptide, which are requirements of other methods. It avoids problems associated with limited proteolysis of an antigen-antibody complex particularly in the vicinity of the binding domain which can hinder identification of the epitope. Epitopic peptides are identified from a direct comparison of the matrix-assisted laser desorption ionization mass spectra of the antibody reaction mixture and an unreacted control. Samples are prepared for mass spectrometric analysis by heat-assisted and electrospray deposition to afford reproducible spectra that enable epitopic peptides to be identified in complex mixtures analyzed at the femtomole level. Indirect evidence is presented to suggest that the antibody-peptide complex is resilient to both sample deposition and the ionization event. The utility and sensitivity of the approach are illustrated for the lysozyme model. IgG-binding domains of human lysozyme are identified, and one epitope is refined to six residues that comprise part of an extended beta-loop region.
本文描述了一种快速的新方法，该方法结合了免疫亲和和基于质谱技术的选择性和灵敏度来绘制蛋白质表位。该方法无需固定抗体、提取抗体 - 肽复合物和解离结合肽，而这些是其他方法的要求。它避免了与抗原 - 抗体复合物有限蛋白水解相关的问题，特别是在结合域附近，这可能会阻碍表位的鉴定。通过直接比较抗体反应混合物和未反应对照的基质辅助激光解吸电离质谱来鉴定表位肽。通过热辅助和电喷雾沉积制备用于质谱分析的样品，以提供可重现的光谱，从而能够在飞摩尔水平分析的复杂混合物中鉴定表位肽。间接证据表明抗体 - 肽复合物对样品沉积和电离事件均具有抗性。该方法的实用性和灵敏度在溶菌酶模型中得到了说明。鉴定了人溶菌酶的IgG结合域，并且将一个表位细化为六个残基，这些残基构成了一个延伸的β-环区域的一部分。
