Biology subjects

Hojrup, P.

Publications and source records attributed to Hojrup, P..

3 recordsLinked to original sources

bioRxiv2026

GPMAW Glyco-Search: An Integrated Workflow for Identification and Validation of Intact Sialylated N-Glycopeptides

Comprehensive analysis of intact sialylated N-glycopeptides remains challenging because of their low abundance, extensive structural heterogeneity, and limited peptide backbone fragmentation during tandem mass spectrometry. Here, we present an integrated workflow for high-confidence identification of intact sialylated N-glycopeptides that combines selective TiO2 enrichment, dual LC-MS/MS analysis of intact and deglycosylated glycopeptides, and the GPMAW glyco-search platform based on high-accuracy mass mapping. Unlike most conventional glycoproteomics search engines, GPMAW uses experimentally identified deglycopeptides to constrain glycan assignment before matching intact glycopeptide precursor masses to candidate glycan compositions. Identifications were validated using diagnostic oxonium ions, glycopeptide-associated Y-ion fragments, and an experimentally derived glycopeptide score. In addition, GPMAW integrates an interactive spectrum annotation interface that enables rapid manual validation of candidate identifications through visualization of annotated Y-ion series, oxonium ions, and peptide fragments, allowing individual assignments to be readily accepted or rejected. The workflow was optimized using bovine fetuin, validated on standard glycoproteins, and applied to depleted human plasma, where more than 2800 unique intact sialylated N-glycopeptides were identified across hundreds of glycosites and glycoproteins. Moreover, more than 1000 unique N-glycopeptides were identified from only 1 L of plasma. Comparative analysis demonstrated that GPMAW glyco-search identified more confidently assigned intact sialylated N-glycopeptides than three widely used N-glycoproteomics search engines while maintaining high reproducibility and low false-positive rates following manual validation. Together, this workflow provides a robust, flexible, and accessible platform for large-scale, high-confidence characterization of intact N-glycopeptides and establishes experimentally constrained glycan composition assignment combined with interactive spectrum validation as an effective strategy for reducing ambiguity in N-glycoproteomics. HighlightsO_LIThe program "GPMAW glyco-search" enables high-accuracy mass mapping for confident identification of intact N-glycopeptides. C_LIO_LIIntegrated workflow combining TiO2 enrichment, dual LC-MS/MS of intact and deglycosylated glycopeptides and GPMAW glyco-search for intact sialylated N-glycopeptides. C_LIO_LIOptimized TiO2 enrichment provides >95% selective enrichment of sialylated N-glycopeptides from complex biological samples. C_LIO_LIInteractive spectrum annotation and Y-ion-based scoring enable rapid manual validation and high-confidence glycopeptide identification. C_LIO_LIGPMAW glyco-search confidently identified more intact sialylated N-linked glycopeptides compared to three established glycoproteomics search engines. C_LI

biochemistry↗

bioRxiv2024

Wild-type and single-O-antigen repeat outer-membrane vesicles induce equivalent protection against homologous and heterologous Salmonella challenge

Lipopolysaccharide O-antigen is an immunodominant target of protective antibodies. Variation in O-antigen structures limits antibody-mediated cross-protection between closely-related pathogens including Salmonella Typhimurium (STm) and S. Enteritidis (SEn). Bacterial outer membrane vesicles (OMV) are vaccine platforms presenting surface antigens in their natural conformations. To assess how O-antigen lengths impact antibody responses and control of homologous or heterologous infection, mice were immunized with STm-OMV containing wild-type O-antigen unit repeats (wt-OMV), [≤]1 O-antigen unit (wzy-OMV), or no O-antigen units (wbaP-OMV) respectively and challenged with either STm or SEn. Unexpectedly, anti-STm LPS IgG and protection to STm were comparable after immunization with either wt-OMV or wzy-OMV. Anti-porin responses were elevated after immunization with wzy-OMV and wbaP-OMV. A single immunization with any OMV induced minimal cross-protection against SEn, except in blood. In contrast, boosting with O-antigen-expressing OMV enhanced control of SEn infections by >10-fold. These results suggest that i) Antibody to single or variable-length O-antigen units are comparably protective against Salmonella; ii) Antigens other than immunodominant O-antigens may be targets of cross-reactive antibodies that moderate bacterial burdens; iii) Boosting can enhance the level of cross-protection against related Salmonella serovars and iv) High tissue burdens of Salmonella can be present in the absence of detectable bacteraemia.

immunology↗

bioRxiv2023

Epitope mapping of SARS-CoV 2 RBDs by hydroxyl radical protein footprinting reveals the importance of including negative antibody controls.

Understanding protein-protein interaction is essential when designing drugs or investigating biological processes. A variety of techniques can be employed in order to map the regions on proteins that are involved in binding eg., CryoEM, X-ray spectroscopy, linear epitope mapping, or mass spectrometry-based methods. The most commonly utilized mass spectrometry-based techniques are cross-linking and hydrogen-deuterium exchange (HDX). An alternative technique for identifying residues on the three-dimensional structure of proteins, that are involved in binding, can be hydroxyl radical protein footprinting (HRPF). However, this method is currently hampered by high initial cost and complex experimental setup. Here we set out to present a generally applicable method using Fenton chemistry for mapping of epitopes in a standard mass spectrometry laboratory. Furthermore, the described method illustrates the importance of controls on several levels when performing mass spectrometry-based epitope mapping. In particular, the inclusion of a negative antibody control has not previously been widely utilized in epitope mapping by HRPF analysis. In order to limit the number of false positives, we further introduced quantification by TMT labelling, thereby allowing for direct comparison between sample conditions and biological triplicates. Lastly, up to six technical replicates were incorporated in the experimental setup in order to achieve increased depth of the final analysis. Both binding and opening of regions on receptor-binding domain (RBD) from SARS-CoV-2 Spike Protein, Alpha, and Delta variants, were observed. The negative control antibody experiment combined with the high overlap between biological triplicates resulted in the exclusion of 40% of the significantly changed regions, including both binding and opening regions. The final identified binding region was mapped to a three-dimensional structure and agrees with the literature for neutralizing antibodies towards SARS-CoV-2 Spike Protein. The presented method is straightforward to implement for the analysis of HRPF in a generic MS-based laboratory. The high reliability of the data was achieved by increasing the number of technical and biological replicates combined with negative antibody controls.

biochemistry↗