Dataset Information

Site-specific Analysis of O-linked Glycoproteome Using Chemoenzymatic Strategy, part 2

ABSTRACT: Protein glycosylation is ubiquitous and plays critical roles in biology. However, study of O-linked glycoproteome (O-glycoproteome), a major type of protein glycosylation, has been severely impeded due to paucity of technology. We presented a chemoenzymatic strategy for extraction of site-specific O-linked glycopeptides (SOGO), which enabled simultaneous enrichment and identification of O-linked glycosylation sites (O-glycosites) in an unprecedented depth. Central to SOGO is a tandem-use of solid-phase capture of peptides and an O-linked glycan (O-glycan) dependent endoprotease named OpeRATOR. Interestingly, OpeRATOR requires O-glycan to specifically cleave the N-terminus of glycan-occupied Ser and Thr leading to unambiguous identification of O-glycosites and corresponding glycoproteins. The identified O-glycosites facilitated downstream determination of microheterogeneity of intact O-linked glycopeptides (O-glycopeptides). We benchmarked the method using human serum and identified 805 peptides containing 777 O-glycosites from 1,345 site-specific O-glycopeptides and 302 glycoproteins. We applied the method to study change of protein-specific O-glycosylation in human T cells infected with human immunodeficiency virus (HIV-1). Strikingly, 1404 peptides containing 1,352 O-glycosites from 577 glycoproteins were identified. In addition, altered site-specific O-linked glycosylation (O-glycosylation) during HIV infection of T cells were observed. In total, 1989 O-glycosites from 789 glycoproteins were precisely pinpointed from human serum and T cells. We observed conserved motifs for O-glycoproteome. Finally, ontology analysis revealed diverse roles for O-glycoproteins arguing broad application of our method to study biology in respective to protein O-glycosylation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood

SUBMITTER: Weiming Yang

LAB HEAD: Hui Zhang

PROVIDER: PXD007895 | Pride | 2022-02-28

REPOSITORIES: Pride

ACCESS DATA

Similar Datasets

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.

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.

Dataset Information

Site-specific Analysis of O-linked Glycoproteome Using Chemoenzymatic Strategy, part 2

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure