Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA.
Protein Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA.
Sci Rep. 2017 Aug 11;7(1):7896. doi: 10.1038/s41598-017-08333-y.
Peptide mapping with liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an important analytical method for characterization of post-translational and chemical modifications in therapeutic proteins. Despite its importance, there is currently no consensus on the statistical analysis of the resulting data. In this manuscript, we distinguish three statistical goals for therapeutic protein characterization: (1) estimation of site occupancy of modifications in one condition, (2) detection of differential site occupancy between conditions, and (3) estimation of combined site occupancy across multiple modification sites. We propose an approach, which addresses these goals in terms of summarizing the quantitative information from the mass spectra, statistical modeling, and model-based analysis of LC-MS/MS data. We illustrate the approach using an LC-MS/MS experiment from an antibody-drug conjugate and its monoclonal antibody intermediate. The performance was compared to a 'naïve' data analysis approach, by using computer simulation, evaluation of differential site occupancy in positive and negative controls, and comparisons of estimated site occupancy with orthogonal experimental measurements of N-linked glycoforms and total oxidation. The results demonstrated the importance of replicated studies of protein characterization, and of appropriate statistical modeling, for reproducible, accurate and efficient site occupancy estimation and differential analysis.
肽图分析与液相色谱-串联质谱（LC-MS/MS）联用是一种用于鉴定治疗性蛋白翻译后修饰和化学修饰的重要分析方法。尽管其重要性不言而喻，但目前对于该类数据的统计分析尚无共识。在本文中，我们将治疗性蛋白的特征描述分为三个统计学目标：（1）在一个条件下估计修饰的位点占有率，（2）检测不同条件下的修饰的位点占有率差异，以及（3）跨多个修饰位点估计修饰的位点占有率的综合值。我们提出了一种方法，该方法通过对质谱的定量信息进行总结、统计建模和基于模型的 LC-MS/MS 数据分析来实现这些目标。我们使用抗体药物偶联物及其单克隆抗体中间物的 LC-MS/MS 实验对该方法进行了说明。通过计算机模拟、阳性和阴性对照中差异修饰的位点占有率评估以及与 N-连接糖型和总氧化的正交实验测量结果的修饰的位点占有率估计值的比较，我们将该方法的性能与“原始”数据分析方法进行了比较。结果表明，对于治疗性蛋白的特征描述重复性研究以及适当的统计建模对于重现性、准确性和高效的修饰的位点占有率估计和差异分析非常重要。
