Hydrophobic-hydration hydrophilic interaction (H3I) capturing intact glycopeptides
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Jing Liu
LAB HEAD: Jing Liu
PROVIDER: PXD033779 | Pride | 2023-04-06
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ACE2_Glyco_TC_HCD-1.raw | Raw | |||
| ACE2_Glyco_TC_HCD-2.raw | Raw | |||
| ACE2_Glyco_TC_HCD-3.raw | Raw | |||
| ACE2_Glyco_TG_HCD-1.raw | Raw | |||
| ACE2_Glyco_TG_HCD-2.raw | Raw |
Items per page: 5 1 - 5 of 21 |
Similar Datasets
Project description:We performed a large-scale, site-specific N-glycoproteome profiling study of human Alzheimer’s disease (AD) and control brains using mass spectrometry–based quantitative N-glycoproteomics. The study provided a system-level view of human brain N-glycoproteins and in vivo N-glycosylation sites and identified disease signatures of altered N-glycopeptides, N-glycoproteins, and N-glycosylation site occupancy in AD. Glycoproteomic data-driven network analysis showed 13 modules of co-regulated N-glycopeptides/glycoproteins, 6 of which are associated with AD phenotypes.
2022-11-02 | PXD037229 | Pride
Project description:Protein N-glycosylation is ubiquitous in brain and closely related to cognition and memory. Alzheimer's disease (AD) is a multifactorial disorder that lacks clear pathogenesis and treatment. Aberrant N-glycosylation has been suggested to be involved in AD pathology. While the systematic variations of protein N-glycosylation and their roles in AD have not been thoroughly investigated due to technically challenging. Here, we applied multilayered N-glycoproteomics to quantify the global protein expression, N-glycosylation sites, N-glycans, and site-specific N-glycopeptides in AD mice brains (APP/PS1 transgenic) versus wild type. The N-glycoproteome landscape exhibited the highly complex site-specific heterogeneity in AD brains. Quantitative analyses explored the generally dysregulated N-glycosylations in AD, involving proteins such as glutamate receptors, as well as fucosylated and oligo-mannose glycans. Furthermore, functional study revealed the crucial roles of N-glycosylation on proteins and neuron cells. Our work provided a robust multilayered N-glycoproteomics workflow for AD and can be applied to widespread biological systems.
2019-11-25 | PXD015335 | Pride
Project description:We have characterized the site-specific glycosylation patterns of the HEK293 recombinant spike RBD and S1 domains as well as the intact spike derived from whole virus produced in Vero cells.
2022-02-16 | PXD029218 | Pride
Project description:Immunoglobulin G (IgG), which contains four subclasses (IgG1-4), is one of the most important class of glycoproteins in immune system. Because of its importance in immune system, a steady increase of interest in developing IgG as biomarker or biotherapeutic agents for the treatment of diseases has been seen, as most therapeutic mAbs were IgG-based. N-glycosylation of IgG is crucial for its effector function and makes the IgG highly heterogeneous both in structure and function, although all four subclasses of IgG contain only a single N-glycosylation site in the Fc region with highly similar amino acid sequence. Therefore, fine mapping the IgG glycosylation is necessary for understanding the IgG function and avoiding aberrant glycosylation in mAbs. However, site-specific and comprehensive N-glycosylation analysis of IgG subclasses still cannot be achieved by MS alone due to the partial sequence coverage and loss of connections among glycosylation on protein sequence. We report here a chemical labeling strategy to improve the electron transfer dissociation efficiency in mass spectrometry analysis, which enable a 100% peptide sequence coverage of N-glycopeptides in all subclasses of IgG. Combining with high-energy collisional dissociation for the fragmentation of glycans, fine mapping of N-glycosylation profile of IgG is achieved. This comprehensive glycosylation analysis strategy for the first time allows the discrimination of IgG3 and IgG4 intact N-glycopeptides with high similarity in sequence without the antibody-based pre-separation. Using this strategy, aberrant serum IgG N-glycosylation for four IgG subclasses associated with cirrhosis and hepatocellular carcinoma were revealed. Moreover, this method identifies 5 times more intact glycopeptides from human serum than native-ETD method, implying that the approach can also accommodate for large-scale site-specific profiling of glycoproteomics.
2019-08-26 | PXD014713 | Pride
Project description:The densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the comprehensive site-specific O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized using high-resolution mass spectrometry. Following digestion using trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the mucin-type (GalNAc-type) O-glycosylation pattern of S proteins, including O-glycosites and the 6 most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 43 O-glycosites were identified by higher energy collision-induced dissociation (HCD), and 11 O-glycosites were verified by electron transfer/higher energy collision-induced dissociation (EThcD) in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O-glycans such as HexNAc(1) and HexNAc(1)Hex(1). In contrast, 30 O-glycosites were identified by HCD, and 14 O-glycosites were verified by EThcD in the human cell-expressed S protein S1 subunit. Most glycosites were modified by sialylated O-glycans such as HexNAc(1)Hex(1)NeuAc(1) and HexNAc(1)Hex(1)NeuAc(2). Our results are the first to reveal that the SARS-CoV-2 S protein is a mucin-type glycoprotein; clustered O-glycans often occur in the N- and the C-termini of the S protein, and the O-glycosite and O-glycan compositions vary with the host cell type. These site-specific O-glycosylation landscapes of the SARS-CoV-2 S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.
2021-11-03 | PXD022896 | Pride
Project description:Identification results of peptide-spectrum matches supporting Glyco-Decipher manuscript (Glyco-Decipher: Glycan database-independent peptide matching enables discovery of new glycans and in-depth characterization of site-specific N-glycosylation). Recently, several elegant bioinformatics tools have been developed to identify glycopeptides from tandem mass spectra for site-specific glycoproteomics studies. These glycan database-dependent tools have substantially improved glycoproteomics analysis but fail to identify glycopeptides with unexpected glycans. We present a platform called Glyco-Decipher to interpret the glycoproteomics data of N-linked glycopeptides. It adopts a glycan database-independent peptide matching scheme that allows the unbiased profiling of glycans and the discovery of new glycans linked with modifications. Reanalysis of several large-scale datasets showed that Glyco-Decipher outperformed the open search method in glycan blind searching and the popular glycan database-dependent software tools in glycopeptide identification. Our glycan database-independent search also revealed that modified glycans are responsible for a large fraction of unassigned glycopeptide spectra in shotgun glycoproteomics.
2022-03-04 | PXD031025 | Pride
Project description:HKU1 is a human betacoronavirus and infects host cells via highly glycosylated spike protein (S). At present, N-glycosylation of HKU1 S has been reported, however, little is known about its O-glycosylation, which hinders an in-depth understanding of its biological functions. Herein, a comprehensive study of O-glycosylation of HKU1 S was carried out based on dual-functional histidine-bonded silica materials (HBS). The enrichment method for O-glycopeptides with HBS was developed and validated using standard proteins. The application of the developed method to HKU1 S1 subunit resulted in 61 novel O-glycosylation sites, among which 56% were predicted to be exposed on protein outer surface. Moreover, the O-linked glycans and their abundance on each HKU1 S1 site were also analyzed. The obtained O-glycosylation dataset would provide valuable insights for the understanding of the structure of HKU1 S.
2022-02-17 | PXD025967 | Pride
Project description:Protein glycosylation is one of the most common protein modifications and plays essential roles in biology and therapeutics. However, the analysis of in vivo O-linked glycosylation, a major type of protein glycosylation, has been severely impeded by the scarcity of technology. Here, a chemoenzymatic method was presented for the site-specific extraction of O-linked glycopeptides (EXoO), which achieved simultaneous enrichment and unambiguous mapping of over 3,000 O-linked glycosylation sites and corresponding O-linked glycans on over 1,000 proteins in human kidney tissues, serum and T cells. The large-scale localization of O-linked glycosylation sites nearly doubles the sites identified in the last decades demonstrating that EXoO is the most effective method to-date for defining the site-specific O-linked glycoproteome in different types of sample. Structural analysis of the sites revealed conserved motifs and topological orientation facing extracellular space or lumen of ER and Golgi. Striking signature of aberrant in vivo O-linked glycoproteome was observed between kidney tumor and normal tissues discovering key factors in tumor biology. The O-linked glycoproteome play diverse roles on the ER, Golgi membrane, cell surface and extracellular space arguing that EXoO can be applied broadly to the analysis of O-linked glycoproteins in biology and therapeutics.
2018-12-05 | PXD009476 | Pride
Project description:We aimed to in-depth characterize and quantify salivary N-glycoproteomics. HILIC enrichment and LC-MS/MS were combined to investigate salivary glycosylation both at deglycopeptides level and glycopepeptides level, and alterations in site-specific glycoforms for human saliva were detected between lung cancer patients and healthy subjects. Firstly, intact glycopeptides were enriched from human saliva through using HILIC. Obtained intact glycopeptides were characterized by LC-MS/MS directly. Furthermore, the glycosylation sites were fully identified by LC-MS/MS followed by incubating with PNGase F. The developed workflow was applied to compare N-glycosites and intact N-glycopeptides in lung cancer group and healthy control group. Dysregulated N-glycosites as well as site-specific glycoforms were confidently observed in lung cancer and their potential value in clinical applications will be discussed.
2023-02-08 | PXD030670 | Pride
Project description:Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans may mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin, and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosites sites were localized by the 18O-labeling method. O-glycopeptide enrichment and O-glycosite identification were achieved by an enzyme-assisted site-specific extraction method. An RP–LC–MS/MS system functionalized with Collision-Induced Dissociation and stepped normalized collision energy (sNCE)-HCD tandem mass was applied to analyze the glycoforms of fibronectin. A total of 6 N-glycosites and 53 O-glycosites were identified, which were occupied by 3842 N-glycoforms and 16 O-glycoforms, respectively. Furthermore, 81.4% of N-glycans were either fucosylated, sialylated, or with both modifications77.31% of N-glycans were sialylated, while O-glycosylation was dominated by the sialyl-T antigen. These site-specific glycosylation patterns on human fibronectin can facilitate functional analyses of fibronectin and therapeutics development.
2022-02-17 | PXD025886 | Pride