Ponniah Gomathinayagam, Nowak Christine, Kita Adriana, Cheng Guilong, Kori Yekaterina, Liu Hongcheng
Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA.
Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA.
Anal Biochem. 2016 Mar 15;497:1-7. doi: 10.1016/j.ab.2015.12.008. Epub 2015 Dec 31.
Limited proteolytic digestion is a method with a long history that has been used to study protein domain structures and conformational changes. A method of combining limited proteolytic digestion, stable isotope labeling, and mass spectrometry was established in the current study to investigate protein conformational changes. Recombinant monoclonal antibodies with or without the conserved oligosaccharides, and with or without oxidation of the conserved methionine residues, were used to test the newly proposed method. All of the samples were digested in ammonium bicarbonate buffer prepared in normal water. The oxidized deglycosylated sample was also digested in ammonium bicarbonate buffer prepared in (18)O-labeled water. The sample from the digestion in (18)O-water was spiked into each sample digested in normal water. Each mixed sample was subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). The molecular weight differences between the peptides digested in normal water versus (18)O-water were used to differentiate peaks from the samples. The relative peak intensities of peptides with or without the C-terminal incorporation of (18)O atoms were used to determine susceptibility of different samples to trypsin and chymotrypsin. The results demonstrated that the method was capable of detecting local conformational changes of the recombinant monoclonal antibodies caused by deglycosylation and oxidation.
有限蛋白酶解是一种历史悠久的方法，已被用于研究蛋白质结构域结构和构象变化。本研究建立了一种将有限蛋白酶解、稳定同位素标记和质谱相结合的方法来研究蛋白质构象变化。使用具有或不具有保守寡糖、以及具有或不具有保守甲硫氨酸残基氧化的重组单克隆抗体来测试新提出的方法。所有样品均在普通水中制备的碳酸氢铵缓冲液中进行消化。氧化去糖基化样品也在（18）O标记水中制备的碳酸氢铵缓冲液中进行消化。将（18）O水消化的样品加入到在普通水中消化的每个样品中。随后对每个混合样品进行液相色谱-质谱（LC-MS）分析。普通水消化的肽与（18）O水消化的肽之间的分子量差异用于区分样品中的峰。具有或不具有（18）O原子C末端掺入的肽的相对峰强度用于确定不同样品对胰蛋白酶和糜蛋白酶的敏感性。结果表明，该方法能够检测去糖基化和氧化引起的重组单克隆抗体的局部构象变化。
