Zhai Ziran, Holmark Thomas, Jasperse Lars, Gargano Andrea F G
Analytical Chemistry Group, Van't Hoff Institute for Molecular Sciences (HIMS), University of Amsterdam, Amsterdam, the Netherlands.
Centre for Analytical Sciences Amsterdam, Van't Hoff Institute for Molecular Sciences (HIMS), University of Amsterdam, Amsterdam, the Netherlands.
J Sep Sci. 2025 Sep;48(9):e70268. doi: 10.1002/jssc.70268.
Ion exchange chromatography (IEC) hyphenated to mass spectrometry (MS) is a powerful non-denaturing technique used for analyzing protein charge heterogeneity (including posttranslational modifications such as deamidation, phosphorylation, and sialylation), as well as for characterizing complex protein mixtures. This review provides an overview of current strategies for implementing IEC-MS, focusing on pH gradient-based methods: chromatofocusing, linear pH gradient elution, and salt-mediated pH gradient elution. First, the fundamental principles, elution modes, and separation mechanisms of IEC are introduced. The review then discusses the limitations of traditional salt gradient elution in IEC, which often relies on nonvolatile additives, and highlights the critical role of pH gradient-based IEC in enabling direct and efficient hyphenation with MS. Key factors influencing protein elution in IEC are summarized to aid method optimization and enhance the understanding of the separation process. Subsequently, recent advancements in IEC-MS are described, covering three key aspects: (i) the selection of appropriate volatile buffer systems and strategies for achieving controlled linear pH gradients, (ii) developments and selection criteria for IEC columns, and (iii) approaches to improve sensitivity and ionization efficiency in IEC-MS. Finally, the review compiles details of recently developed (2010-2025) pH gradient-based IEC-MS methods and summarizes their applications in identifying diverse therapeutic protein variants arising from PTMs, antibody-drug conjugates (ADCs), and other complex biological samples.
与质谱联用的离子交换色谱法（IEC-MS）是一种强大的非变性技术，用于分析蛋白质电荷异质性（包括脱酰胺、磷酸化和唾液酸化等翻译后修饰）以及表征复杂的蛋白质混合物。本综述概述了当前实施IEC-MS的策略，重点关注基于pH梯度的方法：色谱聚焦、线性pH梯度洗脱和盐介导的pH梯度洗脱。首先，介绍了IEC的基本原理、洗脱模式和分离机制。然后，综述讨论了IEC中传统盐梯度洗脱的局限性，其通常依赖于非挥发性添加剂，并强调了基于pH梯度的IEC在实现与MS直接高效联用方面的关键作用。总结了影响IEC中蛋白质洗脱的关键因素，以帮助优化方法并加深对分离过程的理解。随后，描述了IEC-MS的最新进展，涵盖三个关键方面：（i）选择合适的挥发性缓冲系统和实现可控线性pH梯度的策略，（ii）IEC柱的发展和选择标准，以及（iii）提高IEC-MS灵敏度和电离效率的方法。最后，综述汇编了最近开发的（2010-2025年）基于pH梯度的IEC-MS方法的详细信息，并总结了它们在鉴定由翻译后修饰产生的各种治疗性蛋白质变体、抗体药物偶联物（ADC）和其他复杂生物样品中的应用。
