Electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled with mass spectrometry (MS) is a technique that is increasingly being used as a trapping/enrichment tool for glycopeptides/phosphorylated peptides or sample fractionation in proteomics research. Here, we describe a novel ERLIC-MS/MS-based peptide mapping method that was successfully used for the characterization of denosumab, in particular the analysis of sequence coverage, terminal peptides, methionine oxidation, asparagine deamidation and glycopeptides. Compared to reversed phase liquid chromatography (RPLC)-MS/MS methods, ERLIC demonstrated unique advantages in the retention of small peptides, resulting in 100% sequence coverage for both the light and heavy chains. It also demonstrated superior performance in the separation and characterization of asparagine deamidated peptides, which is known to be challenging by RPLC-MS/MS. The developed method can be used alone for peptide mapping-based characterization of monoclonal antibodies, or as an orthogonal method to complement the RPLC-MS/MS method. This study extends the applications of ERLIC from that of a trapping/fractioning column to biologic therapeutics characterization. The ERLIC-MS/MS method can enhance biologic therapeutics analysis with more reliability and confidence for bottom-up peptide mapping-based characterization.

静电排斥亲水相互作用色谱（ERLIC）和质谱（MS）结合使用的技术正越来越多地用作糖蛋白肽/磷酸化肽或蛋白质组学研究中的样品分离的捕集/富集工具。在这里，我们描述了一种新型的基于ERLIC-MS / MS的肽图分析方法，该方法已成功用于denosumab的表征，特别是序列覆盖率，末端肽，蛋氨酸氧化，天冬酰胺脱酰胺和糖肽的分析。与反相液相色谱（RPLC）-MS / MS方法相比，ERLIC在保留小肽方面表现出独特的优势，从而使轻链和重链的序列覆盖率均达到100％。它在天冬酰胺脱酰胺基肽的分离和表征中也表现出优异的性能，众所周知，RPLC-MS / MS具有挑战性。所开发的方法可以单独用于基于肽图的单克隆抗体表征，也可以作为正交方法来补充RPLC-MS / MS方法。这项研究将ERLIC的应用从捕获/分馏柱的应用扩展到了生物学治疗的表征。ERLIC-MS / MS方法可提高生物治疗分析的可靠性和置信度，可用于基于自下而上的肽图鉴定。这项研究将ERLIC的应用从捕获/分馏柱的应用扩展到了生物学治疗的表征。ERLIC-MS / MS方法可提高生物治疗分析的可靠性和置信度，可用于基于自下而上的肽图鉴定。这项研究将ERLIC的应用从捕获/分馏柱的应用扩展到了生物学治疗的表征。ERLIC-MS / MS方法可提高生物治疗分析的可靠性和置信度，可用于基于自下而上的肽图鉴定。