In this study, the effects of four commonly used lysis buffers (sodium dodecyl sulfate (SDS), guanidine hydrochloride (GuHCl), urea (UA), and Mammalian Protein Extraction Reagent (MPER)) on proteome and N-glycosylation modification analysis, providing valuable reference for optimizing experimental workflows in proteomics and glycoproteomics research. Proteins were digested into peptides using the Filter-Aided Sample Preparation (FASP) protocol, followed by enrichment of glycopeptides via Hydrophilic Interaction Liquid Chromatography (HILIC). Proteomic data were analyzed with MaxQuant, while glycoproteomic data were processed using pGlyco3 and Panda software.