조희영 (충남대학교)

안현주 (충남대학교)

조희영 (충남대학교)

정유진 (충남대학교)

김아빈 (충남대학교)

오명진 (충남대학교)

안현주 (충남대학교)

Protein glycosylation is among the most abundant and functionally important post-translational modifications, regulating diverse biological processes including cell-cell communication, immune responses, and receptor-mediated signaling. Mass spectrometry-based glycopeptide (GP) analysis enables the simultaneous characterization of glycan structures, glycosylation sites, and their corresponding glycoproteins, providing protein- and site-specific glycosylation information that is essential for understanding the functional roles of protein glycosylation. However, GP analysis remains technically challenging because analytical performance is highly dependent on sample preparation procedures and nanoLC-MS conditions, resulting in limited reproducibility and analytical consistency. This study presents an analytical strategy for standardizing GP analysis using a nanoLC-Orbitrap mass spectrometry platform. The proposed strategy encompasses analytical steps, with protein digestion, peptide cleanup, glycopeptide enrichment, nanoLC separation, and Orbitrap MS/MS acquisition. Key methodological parameters, including enrichment resin, elution solvent composition, LC gradient length, and collision energy, are systematically optimized. Ongoing analysis of these variables is expected to establish a practical framework for improving the reproducibility, robustness, and consistency of GP analysis and supports the development of standardized workflows for mass spectrometry-based glycoproteomics.