Depletion-free Automated Enrichment of Serum Glycopeptides for High-Throughput Clinical Glycoproteomics.

High-throughput and reproducible enrichment of N-glycopeptides remains a major bottleneck in large-scale and clinical glycoproteomics, despite substantial advances in mass spectrometry. Here, we present a depletion-free, high-throughput N-glycopeptide enrichment workflow (3kMAX) that combines 3 kDa molecular weight cut-off filtration with positive-pressure-driven mixed anion exchange (MAX) enrichment in a 96-well format. This platform enables parallel processing of up to 96 samples with minimal operator intervention, improving analytical throughput, robustness and inter-batch reproducibility. Systematic evaluation using human serum demonstrated that the combined workflow achieved superior glycopeptide selectivity and identification depth compared with conventional manual MAX enrichment or molecular weight cut-off filtration alone, without introducing detectable glycan-type bias. Sensitivity analysis across a fourfold range of serum input volumes (5-20 μl) showed stable identification performance and quantitative reproducibility, with 10 μl identified as an optimal standardized input. Day-to-day reproducibility assessments further confirmed consistent quantitative performance across different acquisition densities. Application of the workflow to hepatocellular carcinoma serum samples enabled reproducible, large-scale quantitative glycoproteomic profiling and revealed extensive disease-associated glycosylation remodeling, including site-specific regulation within individual proteins. Collectively, the 3kMAX platform provides a scalable solution for site-specific serum N-glycoproteomics and facilitates its translation into clinical and biomarker-oriented applications.