18O labeling method for identification and quantification of succinimide in proteins
18O labeling method for identification and quantification of succinimide in proteins
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DOI：
10.1021/ac0617870
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发表时间：
2007-04-01
影响因子：
7.4
通讯作者：
Chelius, Dirk
中科院分区：
文献类型：
--
作者：
Xiao, Gang;Bondarenko, Pavel V.;Chelius, Dirk
建立了一种新的蛋白质中琥珀酰亚胺的鉴定和定量方法。该方法利用O-18水来监测琥珀酰亚胺的水解。O-18标记的异天冬氨酸和天冬氨酸肽通过在O-18水（（H2O）-O-18）中水解含琥珀酰亚胺的蛋白质，然后在常规水（（H2O）-O-16）中进行胰蛋白酶消化而产生。掺入O-18的肽比其O-16天然对应物重2 Da。通过电喷雾飞行时间质谱法检测和定量质量差异。将O-18掺入含异天冬氨酸和含天冬氨酸肽中的量用于定量天然样品中存在的琥珀酰亚胺的量。该方法被应用于分析降解的重组单克隆抗体，其表现出在温和的酸性缓冲液中在高温下储存数周后琥珀酰亚胺的积累。我们明确鉴定了位于抗体轻链中的氨基酸残基30为天冬氨酸异构化位点。在该位点，降解样品（8周，45 ℃，pH 5.0）中通过肽图谱检测到20%异天冬氨酸和80%天冬氨酸。在O-18水中的水解表明，80%的异天冬氨酸和6%的天冬氨酸具有O-18掺入。O-18掺入的唯一解释是样品中存在琥珀酰亚胺。总之，发现在该降解样品中共有21%（0.8 x 20%异天冬氨酸+0.06 x 80%天冬氨酸）的天冬氨酸残基30以琥珀酰亚胺形式存在。作为对照，使用常规O-16水分析的相同样品未显示任何O-18水的掺入。通过在pH 8.2的变性和天然条件下随时间监测O-18标记的异天冬氨酸和天冬氨酸的量，我们发现，在变性条件下，轻链残基30处的琥珀酰亚胺水解非常迅速（小于5 s），但在天然条件下水解较慢（琥珀酰亚胺半衰期类似于6 h）。我们还发现，在变性条件下，琥珀酰亚胺水解的异天冬氨酸/天冬氨酸的比例为3.5：1，但水解几乎完全天冬氨酸在天然条件下。这一发现表明，蛋白质结构起着重要的作用，在琥珀酰亚胺水解的动力学，以及在水解产物异天冬氨酸和天冬氨酸的产生。
We have developed a new method for identification and quantification of succinimide in proteins. The method utilizes O-18 water to monitor succinimide hydrolysis. O-18-labeled isoaspartic acid and aspartic acid peptides were produced by hydrolysis of a succinimide-containing protein in O-18 water ((H2O)-O-18) followed by tryptic digestion in regular water ((H2O)-O-16). The peptides that had O-18 incorporated were 2 Da heavier than their O-16 native counterparts. The mass difference was detected and quantified by electrospray time-of-flight mass spectrometry. The amount of O-18 incorporation into the isoaspartic acid- and aspartic acid-containing peptides was used to quantify the amount of succinimide present in the native sample. The method was applied to analyze a degraded recombinant monoclonal antibody, which exhibited the accumulation of succinimide after storage in mildly acidic buffers at elevated temperatures for a few weeks. We unambiguously identified amino acid residue 30 located in the antibody light chain as the site of aspartic acid isomerization. At this site, there were 20% isoaspartic acid and 80% aspartic acid detected by peptide mapping in the degraded sample (8 weeks, 45 degrees C, pH 5.0). Hydrolysis in O-18 water showed that 80% of the isoaspartic acid and 6% of the aspartic acid had O-18 incorporated. The only explanation of O-18 incorporation was the presence of succinimide in the sample. Together, a total of 21% (0.8 x 20% isoaspartic acid + 0.06 x 80% aspartic acid) of aspartic acid residue 30 was found to be present in the form of succinimide in this degraded sample. As a control, the same sample, analyzed using regular O-16 water did not show any incorporation of O-18 water. By monitoring the amount of O-18-labeled isoaspartic acid and aspartic acid over time under both denaturing and native conditions at pH 8.2, we found that, at denaturing conditions, succinimide at light chain residue 30 hydrolyzed very rapidly (in less than 5 s), but slower (succinimide half-life of similar to 6 h) under native conditions. We also found that, under denaturing conditions, succinimide hydrolyzed at an isoaspartic acid/aspartic acid ratio of 3.5:1, but hydrolyzed almost exclusively to aspartic acid under native conditions. This finding indicates that protein structure plays an important role in the kinetics of succinimide hydrolysis as well as in the generation of the hydrolysis products isoaspartic acid and aspartic acid.