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Development of an LC-MS/MS peptide mapping protocol for the NISTmAb
Analytical and Bioanalytical Chemistry ( IF 4.3 ) Pub Date : 2018-02-07 , DOI: 10.1007/s00216-018-0848-6
Trina Mouchahoir , John E. Schiel

Peptide mapping is a component of the analytical toolbox used within the biopharmaceutical industry to aid in the identity confirmation of a protein therapeutic and to monitor degradative events such as oxidation or deamidation. These methods offer the advantage of providing site-specific information regarding post-translational and chemical modifications that may arise during production, processing or storage. A number of such variations may also be induced by the sample preparation methods themselves which may confound the ability to accurately evaluate the true modification levels. One important focus when developing a peptide mapping method should therefore be the use of sample preparation conditions that will minimize the degree of artificial modifications induced. Unfortunately, the conditions that are amenable to effective reduction, alkylation and digestion are often the same conditions that promote unwanted modifications. Here we describe the optimization of a tryptic digestion protocol used for peptide mapping of the NISTmAb IgG1κ which addresses the challenge of balancing maximum digestion efficiency with minimum artificial modifications. The parameters on which we focused include buffer concentration, digestion time and temperature, as well as the source and type of trypsin (recombinant vs. pancreatic; bovine vs porcine) used. Using the optimized protocol we generated a peptide map of the NISTmAb which allowed us to confirm its identity at the level of primary structure.

Open image in new windowGraphical abstract
Graphical abstract

Peptide map of the NISTmAb RM 8671 monoclonal antibody. Tryptic digestion was performed using an optimized protocol and followed by LC-UV-MS analysis. The trace represents the total ion chromatogram. Each peak was mapped to peptides identified using mass spectrometry data.



中文翻译:

为NISTmAb开发LC-MS / MS肽图分析方案

肽图分析是生物制药工业中使用的分析工具箱的一部分,可帮助确认蛋白质治疗剂的身份并监测降解事件,例如氧化或脱酰胺。这些方法的优点是可以提供有关在生产,加工或存储过程中可能发生的翻译后修饰和化学修饰的特定位置信息。样品制备方法本身也可能引起许多此类变化,这可能会混淆准确评估真实修饰水平的能力。因此,开发肽图分析方法时,一个重要的重点应该是使用样品制备条件,该条件将使人工修饰的程度降到最低。不幸的是,这些条件可以有效减少,烷基化和消化通常是促进不想要的修饰的相同条件。在这里,我们描述了用于NISTmAbIgG1κ肽图分析的胰蛋白酶消化方案的优化,该方案解决了平衡最大消化效率和最少人工修饰的挑战。我们关注的参数包括缓冲液浓度,消化时间和温度,以及所用胰蛋白酶的来源和类型(重组与胰腺;牛与猪)。使用优化的方案,我们生成了NISTmAb的肽图,这使我们能够在一级结构水平上确认其身份。在这里,我们描述了用于NISTmAbIgG1κ肽图分析的胰蛋白酶消化方案的优化,该方案解决了平衡最大消化效率和最少人工修饰的挑战。我们关注的参数包括缓冲液浓度,消化时间和温度,以及所用胰蛋白酶的来源和类型(重组与胰腺;牛与猪)。使用优化的方案,我们生成了NISTmAb的肽图,这使我们能够在一级结构水平上确认其身份。在这里,我们描述了用于NISTmAbIgG1κ肽图分析的胰蛋白酶消化方案的优化,该方案解决了平衡最大消化效率和最少人工修饰的挑战。我们关注的参数包括缓冲液浓度,消化时间和温度,以及所用胰蛋白酶的来源和类型(重组与胰腺;牛与猪)。使用优化的方案,我们生成了NISTmAb的肽图,这使我们能够在一级结构水平上确认其身份。

在新窗口中打开图像图形概要
图形概要

NISTmAb RM 8671单克隆抗体的肽图。使用优化方案进行胰蛋白酶消化,然后进行LC-UV-MS分析。迹线代表总离子色谱图。将每个峰映射到使用质谱数据鉴定的肽。

更新日期:2018-02-07
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