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Site-specific N-glycoproteomics of aging mouse uterus tissues for benchmarking the StrucGAP analysis platform

ABSTRACT: This project presents the site-specific N-glycoproteomics dataset of aging mouse uterus tissues used to demonstrate and validate StrucGAP, a modular and scalable downstream analysis platform for glycoproteomics. Uterine samples were collected from 2- and 12-month-old C57BL/6 female mice (n = 15 per group), followed by protein extraction, tryptic digestion, TMT 10-plex labeling, intact glycopeptide enrichment, and LC-MS/MS analysis. A total of 20,251 unique intact glycopeptides (IGPs) were identified using the StrucGP search engine. The dataset captures detailed structural and quantitative glycosylation patterns across aging, enabling downstream analyses of glycan substructures, glycosylation site variation, and functional glycoproteomic annotation.

ORGANISM(S): Mus Musculus

SUBMITTER: Shisheng Sun

PROVIDER: PXD063260 | iProX | Wed Apr 23 00:00:00 BST 2025

REPOSITORIES: iProX

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Project description:STT3A and STT3B are the main catalytic subunit of oligosaccharyltransferase (OST-A and OST-B in mammalian cells), which primarily mediate cotranslational and posttranslocational N-linked glycosylation, respectively. To determine the specificity of STT3A and STT3B, we performed proteomics and glycoproteomics analyses to characterize the protein expression and glycosylation in STT3A-knockout (KO), STT3B-KO, and wild-type (WT) HEK293 cells. In total, 3,993 proteins, 4,020 unique N-linked intact glycopeptides (IGPs) and 723 glycosites, representing 401 glycoproteins were identified in KO and WT cells. Deletion of the STT3A gene had a greater impact on the protein expression than deletion of STT3B, especially on glycoproteins. In addition, mannosylated N-glycans from glycoproteins were reduced, while fucosylated N-glycans were increased in STT3A-KO cells. The glycan changes may be caused by the differential expression of glycosyltransferases and the activation of unfolded protein response (UPR) with further analysis of glycan-related enzymes. Interestingly, hyperglycosylated proteins were identified in KO cells. We verified that the hyperglycosylation of ENPL was caused by the ER stress and UPR, which were induced by the deletion of the STT3A. Overall, the specificity of STT3A and STT3B revealed that defects in the OST subunit not only broadly affect N-linked glycosylation of the protein but also affect protein expression.STT3A and STT3B are the main catalytic subunit of oligosaccharyltransferase (OST-A and OST-B in mammalian cells), which primarily mediate cotranslational and posttranslocational N-linked glycosylation, respectively. To determine the specificity of STT3A and STT3B, we performed proteomics and glycoproteomics analyses to characterize the protein expression and glycosylation in STT3A-knockout (KO), STT3B-KO, and wild-type (WT) HEK293 cells. In total, 3,993 proteins, 4,020 unique N-linked intact glycopeptides (IGPs) and 723 glycosites, representing 401 glycoproteins were identified in KO and WT cells. Deletion of the STT3A gene had a greater impact on the protein expression than deletion of STT3B, especially on glycoproteins. In addition, mannosylated N-glycans from glycoproteins were reduced, while fucosylated N-glycans were increased in STT3A-KO cells. The glycan changes may be caused by the differential expression of glycosyltransferases and the activation of unfolded protein response (UPR) with further analysis of glycan-related enzymes. Interestingly, hyperglycosylated proteins were identified in KO cells. We verified that the hyperglycosylation of ENPL was caused by the ER stress and UPR, which were induced by the deletion of the STT3A. Overall, the specificity of STT3A and STT3B revealed that defects in the OST subunit not only broadly affect N-linked glycosylation of the protein but also affect protein expression.

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Site-specific N-glycoproteomics of aging mouse uterus tissues for benchmarking the StrucGAP analysis platform

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