Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

SARS-CoV-2 spike protein glycosylation enriched by HILIC and Ti-IMAC

ABSTRACT: In this work, we performed a fully descriptive analysis N- and O- linked glycosylation of SARS-COV-2 S glycoprotein. We investigated that dual-functionalized Ti-IMAC material enable the simultaneous enrichment and separation of neutral and sialyl glycopeptides of a recombinant SARS-CoV-2 S glycoprotein from HEK293, which will eliminate the signal suppression of neutral glycopeptides to sialyl glycopeptides and improve the glycoform coverage of S protein. We have profiled 19 of its 22 potential N-glycosylated sites with 398 unique glycoforms in dual-functional Ti-IMIAC approach that is 1.6-fold of that in conventional HILIC method. We also identified O-linked glycosylation site that was not found in dual-functional Ti-IMIAC approach. In addition, we have also identified mannose-6-phosphate (M6P) glycosylation, which substantially expands the current knowledge of the spike protein’s glycosylation and enables the investigation of the influence of mannose-6-Phosphate on its cell entry.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Junfeng Huang

LAB HEAD: Lingjun Li

PROVIDER: PXD026450 | Pride | 2022-01-11

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	20201208_SARS_COV_2_S_Ty_CTy_HILIC_HCD_1.byrslt	Other
	20201208_SARS_COV_2_S_Ty_CTy_TiIMAC_FA_HCD_1.byrslt	Other
	20201208_SARS_COV_2_S_Ty_CTy_TiIMAC_TFA_HCD_1.byrslt	Other
	20201208_S_HILIC_2_0_5ug_1.raw	Raw
	20201208_S_HILIC_2_0_5ug_1_Byonic_results.xlsx	Xlsx

Items per page:

1 - 5 of 9

Similar Datasets

glyXtoolMS Example Data Sets - glyXtoolMS: An Open-Source Pipeline for Semiautomated Analysis of Glycopeptide Mass Spectrometry Data

Project description:The data sets of human IgG and human fibrinogen are used as test data for the publication of glyXtoolMS (https://github.com/glyXera/glyXtoolMS). GlyXtoolMS is an open-source analysis software for the (semi)automated targeted analysis of glycopeptide mass spectrometry data using OpenMS/TOPPAS as a framework and pipeline engine. The proteins were selected to show the successful analysis of both N-glycopeptide and O-glycopeptide samples. The samples were measured by nano reversed phase liquid chromatography coupled online to an electrospray ionization orbitrap mass spectrometer (nano RP-LC ESI- OT-MS/MS; LTQ Orbitrap Elite, Thermo Scientific, Waltham, MA, USA) with HCD fragmentation.

2018-09-24 | PXD009716 | Pride

Mouse lung N-glycoproteome, phosphoproteome, and M6P glycoproteome

Project description:This is the raw data and Byonic search results of mouse mouse lung N-glycoproteome, phosphoproteome, and Mannose-6-Phosphate glycoproteome.

2022-01-11 | PXD020196 | Pride

Boost-DiLeu: Enhanced Isobaric N,N-Dimethyl Leucine Tagging Strategy for Comprehensive Quantitative Glycoproteomic Analysis

Project description:Intact glycopeptide analysis has been of great interest because it can elucidate glycosylation site information and glycan structural composition at the same time. However, mass-spectrometry (MS)-based glycoproteomic analysis is hindered by the low stoichiometry of glycosylation and poor ionization efficiency of glycopeptides. Due to the relatively large amounts of starting materials needed for enrichment, identification and quantification of intact glycopeptides in some size-limited biological systems are especially challenging. To overcome these limitations, here we developed an improved boosting strategy to enhance N,N-dimethyl leucine tagging-based quantitative glycoproteomic analysis, termed as Boost-DiLeu. With integration of one-tube sample processing workflow and high pH fractionation, 3514 quantifiable N-glycopeptides were identified from 30 µg HeLa cell tryptic digests with reliable quantification performance. Furthermore, this strategy was applied to human cerebrospinal ﬂuid (CSF) samples to differentiate N-glycosylation profiles between Alzheimer’s disease patients and healthy donors. The results revealed processes and pathways affected by dysregulated N-glycosylation in AD, including platelet degranulation, cell adhesion, and extracellular matrix, which highlighted the involvement of N-glycosylation aberrations in AD pathogenesis. Moreover, co-expression network analysis (WGCNA) showed 3 modules of glycoproteins, one of which are associated with AD phenotype. Our results demonstrated the feasibility of using this strategy for comprehensive glycoproteomic analysis of size-limited clinical samples. Taken together, we developed and optimized a strategy for enhanced comprehensive quantitative intact glycopeptide analysis with DiLeu labeling, which is especially promising for identifying novel therapeutic targets or biomarkers in sample size limited models or systems.

2022-09-08 | PXD029269 | Pride

Finding the Sweet Spot in ERLIC Mobile Phase for Simultaneous Enrichment of N-Glyco and Phosphopeptides

Project description:Simultaneous enrichment of glyco- and phospho- peptides will benefit the studies of biological processes regulated by these post-translational modifications (PTMs). It will also reveal potential crosstalk between these two ubiquitous PTMs. Unlike custom designed multifunctional solid phase extraction (SPE) materials, operating strong-anion exchange (SAX) resin in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) mode provides a readily available strategy to analytical labs for enrichment of these PTMs for subsequent mass spectrometry (MS)-based characterization. However, the choice of mobile phase has largely relied on empirical rules from hydrophilic interaction chromatography (HILIC) or ion-exchange chromatography (IEX) without further optimization and adjustments. In this study, ten mobile phase compositions of ERLIC were systematically compared; the impact of multiple factors including organic phase proportion, ion pairing reagent, pH and salt on the retention of glycosylated and phosphorylated peptides were evaluated. This study demonstrated good enrichment of glyco- and phospho- peptides from the nonmodified peptides in a complex tryptic digest. Moreover, the enriched glyco- and phospho- peptides elute in different fractions by orthogonal retention mechanisms of hydrophilic interaction and electrostatic interaction in ERLIC, maximizing the LC-MS identification of each PTM. The optimized mobile phase can be adapted to the ERLIC HPLC system, where the high resolution in separating multiple PTMs will benefit large-scale MS-based PTM profiling and in-depth characterization.

2019-09-06 | PXD015179 | Pride

An integrated strategy reveals complex glycosylation of erythropoi-etin using top-down and bottom-up mass spectrometry

Project description:The characterization of therapeutic glycoproteins is challenging due to the structural micro- and macro-heterogeneity of the protein glycosylation. This study presents an in-depth strategy for glycosylation analysis using first-generation erythropoietin (epoetin beta), including a developed top-down mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation and a LC-MS approach for O-glycan identi-fication. Permethylated N-glycans, peptides and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). Extending the coverage of our newly developed Python script to phosphorylated N-glycans enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin. The site-specificity of N-glycans was revealed at glycopeptide level by pGlyco software using different proteases. In total, 215 N-glycan compositions were identified from N-glycan and glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two different O-glycan compositions, based on the mass shifts between non-O-glycosylated and O-glycosylated species. This integrated strategy allows the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

2021-06-14 | PXD024835 | Pride

Urinary glycopeptide analysis for the investigation of novel biomarkers

Project description:The aim of this study was to investigate the presence of intact O- and N-glycopeptides in profiles of normal human urine analyzed by capillary electrophoresis coupled to mass spectrometry (CE-MS).CE and liquid chromatography coupled to tandem mass spectrometry (CE- and LC-MS/MS) were both used to identify glycopeptide sequences. Furthermore, we estimated the abundance levels of these glycopeptides in urine from five different cancer types i.e. bladder cancer (BCa), prostate cancer (PCa), pancreatic cancer, cholangiocarcinoma (CC) and renal cell carcinoma (RCC).

2018-11-29 | PXD011214 | Pride

Glycoproteomic and Proteomic analysis of Burkholderia cenocepacia reveals glycosylation events within FliF and MotB are dispensable for motility. - HILIC enriched samples

Project description:Within the Burkholderia genus O-linked protein glycosylation is now known to be highly conserved at the pathway and glycosylation substrate levels. While inhibition of glycosylation has been shown to be detrimental to virulence in B. cenocepacia, little is known about the role of glycosylation in Burkholderia glycoproteins. Within this study we have sought to improve our understanding of the breadth and dynamics of the B. cenocepacia O-glycoproteome to identify glycoproteins which require glycosylation for functionality. Assessing the glycoproteome across multiple common culturing media (LB, TSB, and artificial sputum medium to simulate cystic fibrosis sputum-like conditions) we demonstrate at least 141 glycoproteins are subjected to glycosylation within B. cenocepacia K56-2. Leveraging this insight, we quantitively assessed the glycoproteome of B. cenocepacia using Data-Independent Acquisition (DIA) across culturing media and growth phases revealing most B. cenocepacia glycoproteins are express under all conditions. Examination of how the absence of glycosylation impacts the glycoproteome reveals only a subset of the glycoproteome (BCAL1086, BCAL2974, BCAL0525, BCAM0505 and BCAL0127) appear impacted by the loss of glycosylation. Assessing the proteomic and phenotypic impacts of the loss of these glycoproteins compared to glycosylation null strains revealing the loss of BCAL0525, and to a lesser extend BCAL0127, mirror the proteomic effects observed in the absence of glycosylation. Finally, we demonstrate the loss of glycosylation within BCAL0525 at Serine-358 results in both loss of motility and proteomic impacts on flagellar apparatus consistent with the loss of apparatus stability. Combined this work demonstrates that O-linked glycosylation of BCAL0525 is functionally important within B. cenocepacia.

2024-04-17 | PXD043194 | Pride

Analysis of Pancreatic Extracellular Matrix Protein Post-Translational Modifications via Electrostatic Repulsion-Hydrophilic Interaction Chromatography Coupled with Mass Spectrometry

Project description:The pancreas is a vital organ with digestive and endocrine roles, and diseases of the pancreas affect millions of people yearly. A better understanding of the pancreas proteome and its dynamic post-translational modifications (PTMs) is necessary to engineer higher fidelity tissue analogues for use in transplantation. The extracellular matrix (ECM) has major roles in binding and signaling essential to the viability of insulin-producing islets of Langerhans. To analyze post-translational modifications (PTMs) in the pancreas, native and decellularized tissues from four donors were analyzed.

2021-08-06 | PXD025048 | Pride

ATP-Coated Dual-functionalized Titanium (IV) IMAC Material for Simultaneous Enrichment and Separation of Glycopeptides and Phosphopeptides

Project description:Protein glycosylation and phosphorylation are two of the most common post-translational modifications (PTMs), which plays an important role in many biological processes. However, low abundance and poor ionization efficiency of phosphopeptides and glycopeptides make direct MS analysis challenging. Previously, we explored the electrostatic and hydrophilic properties of commercial centrifuge-assisted-extraction Titanium (IV) IMAC (CAE-Ti-IMAC) material and its application in simultaneously enriching and separating common glycopeptides, phosphopeptides, and M6P glycopeptides in dual-mode. In this study, we developed a hydrophilicity enhanced dual-functional Ti-IMAC material with adenosine triphosphate as grafted group (denoted as: epoxy-ATP-Ti4+) to achieve better enrichment performance in dual-mode separation. The epoxy-ATP-Ti4+ IMAC material was prepared from commercially available epoxy functionalized silica particles in a facile way, which only required two steps of reaction. The ATP molecule not only provided superiorly strong and active metal phosphate sites to bind phosphopeptides, but also contributed significantly to the hydrophilicity to enrich glycopeptides. The epoxy-ATP-Ti4+ IMAC material showed great selectivity and sensitivity for phosphopeptide enrichment in conventional IMAC mode. With optimized buffer and fractionation, the material successfully separated glycopeptides and phosphopeptides with high specificity. Besides standard protein samples, the material was further applied to HeLa cells and mouse lung tissue samples. Our method allows simple and effective enrichment and separation of glycopeptides and phosphopeptides, which paves the way for studying the potential crosstalk between these two PTMs.

2023-07-20 | PXD029775 | Pride

glL enzymes of Burkholderia species are serine preferring oligosaccharidetransferases which targets conserved proteins and sites across the genus- glycoproteomics analysis

Project description:Glycoproteomic analyis of B. cenocepacia strains, K56-2 and H111. ZIC-HILC Glycopeptide enrichment of Trypsin, Thermolysin and Pepsin digested samples.

2021-07-21 | PXD024090 | Pride