Recent advancements in sample pretreatment methods for glycoproteomics

Comprehensive glycoprotein analysis is essential for exploring the role of protein glycosylation in diverse biological processes and disease mechanisms. Yet it remains challenging due to the structural complexity and heterogeneity of glycans. Bottom-up glycoproteomics utilizing liquid chromatography-mass spectrometry (LC-MS)-based techniques has emerged as a powerful tool for in-depth protein glycosylation analysis. Sample pretreatment is the first and critical step that significantly influences subsequent chromatographic separation and MS analysis. This review provides an overview of the key steps in current sample pretreatment workflows for glycoproteomics, emphasizing recent advancements in sample preparation and enrichment strategies developed over the past decade. It highlights improvements in enrichment efficiency, compatibility with highthroughput analyses, and applications to biological samples, and also discusses the remaining challenges and future directions for these technologies.