MOBILion Systems, Inc., 4 Hillman Drive, Suite 130, Chadds Ford, Pennsylvania 19317, United States.
Agilent Technologies Inc., 5301 Stevens Creek Bouelvard, Santa Clara, California 95051, United States.
J Am Soc Mass Spectrom. 2021 Aug 4;32(8):2019-2032. doi: 10.1021/jasms.0c00434. Epub 2021 Apr 9.
Characterization and monitoring of post-translational modifications (PTMs) by peptide mapping is a ubiquitous assay in biopharmaceutical characterization. Often, this assay is coupled to reversed-phase liquid chromatographic (LC) separations that require long gradients to identify all components of the protein digest and resolve critical modifications for relative quantitation. Incorporating ion mobility (IM) as an orthogonal separation that relies on peptide structure can supplement the LC separation by providing an additional differentiation filter to resolve isobaric peptides, potentially reducing ambiguity in identification through mobility-aligned fragmentation and helping to reduce the run time of peptide mapping assays. A next-generation high-resolution ion mobility (HRIM) technique, based on structures for lossless ion manipulations (SLIM) technology with a 13 m ion path, provides peak capacities and higher resolving power that rivals traditional chromatographic separations and, owing to its ability to resolve isobaric peptides that coelute in faster chromatographic methods, allows for up to 3× shorter run times than conventional peptide mapping methods. In this study, the NIST monoclonal antibody IgG1κ (NIST RM 8671, NISTmAb) was characterized by LC-HRIM-MS and LC-HRIM-MS with collision-induced dissociation (HRIM-CID-MS) using a 20 min analytical method. This approach delivered a sequence coverage of 96.5%. LC-HRIM-CID-MS experiments provided additional confidence in sequence determination. HRIM-MS resolved critical oxidations, deamidations, and isomerizations that coelute with their native counterparts in the chromatographic dimension. Finally, quantitative measurements of % modification were made using only the /-extracted HRIM arrival time distributions, showing good agreement with the reference liquid-phase separation. This study shows, for the first time, the analytical capability of HRIM using SLIM technology for enhancing peptide mapping workflows relevant to biopharmaceutical characterization.
肽图分析是生物制药分析中常用的一种检测翻译后修饰（PTMs）的方法。通常，该方法与反相液相色谱（LC）分离相结合，需要长梯度来鉴定蛋白质消化物的所有成分，并解析相对定量的关键修饰。将离子淌度（IM）作为一种依赖于肽结构的正交分离方法，可以通过提供额外的区分滤波器来分离等电点肽，从而补充 LC 分离，这有助于解决鉴定中的歧义，并有助于减少肽图分析检测的运行时间。基于无损离子操控结构（SLIM）技术的新一代高分辨率离子淌度（HRIM）技术，具有 13 米的离子路径，提供了与传统色谱分离相媲美的峰容量和更高的分辨率，并且由于其能够分离在更快的色谱方法中共洗脱的等电点肽，与传统的肽图分析方法相比，运行时间可缩短至原来的 1/3。在这项研究中，使用 20 分钟的分析方法，通过 LC-HRIM-MS 和 LC-HRIM-MS 与碰撞诱导解离（HRIM-CID-MS）对 NIST 单克隆抗体 IgG1κ（NIST RM 8671，NISTmAb）进行了表征。这种方法实现了 96.5%的序列覆盖率。LC-HRIM-CID-MS 实验为序列测定提供了更多的可信度。HRIM-MS 解析了在色谱维度上与天然产物共洗脱的关键氧化、脱酰胺和异构化修饰。最后，仅使用 HRIM 到达时间分布的/-提取进行定量测量，与参考液相分离结果吻合良好。本研究首次展示了使用 SLIM 技术的 HRIM 用于增强与生物制药分析相关的肽图分析工作流程的分析能力。
