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Depletion-Free Automated Enrichment of Plasma Glycopeptides for High-Throughput and Comprehensive Glycoproteomic Profiling

ABSTRACT: We developed 3kMAX, a depletion-free and fully automated N-glycopeptide enrichment platform combining 3 kDa MWCO filtration and positive-pressure MAX enrichment in a 96-well format. It processes up to 96 serum samples with minimal hands-on time and improves glycopeptide selectivity, identification depth, and quantitative reproducibility. Benchmarking confirmed robust, unbiased performance across inputs and throughputs. Applied to hepatocellular carcinoma serum, 3kMAX enabled high-throughput site-specific glycoproteomics and revealed disease-associated glycosylation remodeling.

INSTRUMENT(S): Orbitrap Astral Zoom

ORGANISM(S): Human

SUBMITTER: Jin Young Kim

PROVIDER: MSV000100835 | MassIVE | Fri Feb 13 05:30:00 GMT 2026

SECONDARY ACCESSION(S): PXD074428

REPOSITORIES: MassIVE

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Project description:Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) in human serum is challenging due to the wide dynamic range of serum protein abundances, the lack of a complete serum N-Glycan database and the existence of non-specifically digested peptides and numerous modifications. In this regard, a spectral library search method was presented for serum N-linked intact glycopeptides identification with target-decoy and motif-specific false discovery rate (FDR) control. Low-abundant glycoproteins were firstly separated from high-abundant proteins by acetonitrile (ACN) precipitation. After digestion, the N-linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) column and a portion of the enriched N-linked intact glycopeptides were processed by N-Glycosidase F (PNGase F) to generate de-glycopeptides. Both N-linked intact glycopeptides and de-glycopeptides were analyzed by LC-MS/MS. From N-linked de-glycopeptides datasets, 764 N-linked glycoproteins, 1,699 N-linked glycosites and 3,328 unique N-linked de-glycopeptides were identified. The spectra of these N-linked de-glycopeptides were utilized for N-linked de-glycopeptides library construction and identification of N-linked intact glycopeptides. Four types of N-linked glycosylation motifs (NXS/T/C/V, X≠P) were used to recognize the N-linked de-glycopeptides, and two different N-Glycan mass databases including 27 modified N-Glycan masses and 712 unmodified N-Glycan masses were combined and utilized during spectral library search for identification of N-linked intact glycopeptides. In total, from the N-linked intact glycopeptides datasets, 526 N-linked glycoproteins, 1,036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-Glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at N-linked intact glycopeptide level and N-linked de-glycopeptides level.

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Depletion-Free Automated Enrichment of Plasma Glycopeptides for High-Throughput and Comprehensive Glycoproteomic Profiling

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