Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Human Breast LC-MSMS - Quantitative site- and structure-specific N-glycoproteomics characterization of differential N-glycosylation in MCF-7/ADR cancer stem cells

ABSTRACT: Cancer stem cells (CSCs) are reported to be responsible for tumor initiation, progression, metastasis, and therapy resistance where P-glycoprotein (P-gp) as well as other glycoproteins are involved. Identification of these glycoprotein markers is critical for understanding the resistance mechanism and developing therapeutics. Here we report our comparative N-glycoproteomics study of MCF‐7/ADR vs. MCF-7 cells. With zic-HILIC enrichment, isotopic diethyl labeling, RPLC-MS/MS (HCD) analysis and GPSeeker DB search, differentially expressed N-glycosylation was quantitatively characterized at the intact N-glycopeptides levels.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Stem Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Yue Wang

LAB HEAD: Zhixin Tian

PROVIDER: PXD013836 | Pride | 2020-02-21

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Publications

Quantitative site- and structure-specific N-glycoproteomics characterization of differential N-glycosylation in MCF-7/ADR cancer stem cells.

Xu Feifei F Wang Yue Y Xiao Kaijie K Hu Yechen Y Tian Zhixin Z Chen Yun Y

Clinical proteomics 20200205

<h4>Background</h4>Cancer stem cells (CSCs) are reported to be responsible for tumor initiation, progression, metastasis, and therapy resistance where P-glycoprotein (P-gp) as well as other glycoproteins are involved. Identification of these glycoprotein markers is critical for understanding the resistance mechanism and developing therapeutics.<h4>Methods</h4>In this study, we report our comparative and quantitative site- and structure-specific N-glycoproteomics study of MCF-7/ADR cancer stem ce ...[more]

PMID: 32042278

Similar Datasets

Putative N-glycoprotein markers of MCF-7/ADR cancer stem cells from N-glycoproteomics characterization of the whole cell lysate

Project description:Being the most malignant disease among females, breast cancer has morbidity and mortality in the first and second positions among various cancers [1]. Up to now, chemotherapy has been the most common therapy [2]; however, multidrug resistance (MDR) often occurs to give low overall survival rate. Thus, it is urgent to figure out the mechanism of chemotherapy resistance. The first step of drugs to attack tumor cells is to interact with the plasma membrane, and solute carrier (SLC) family proteins are uptake transporters [3]. On the other side, the efflux transporters (such as ATP-binding cassette, ABC) pump drug out of cancer cells [4]. The main mechanism of MDR is the upregulation of ATP-binding cassette (ABC) transporters [5]. Breast cancer resistance protein (ABCG2), multidrug resistance protein (ABCC1) and P-glycoprotein (P-gp) have been extensively studied [6,7]. Formation of CSCs [8], DNA damage [9] and cell apoptosis [10] and epithelial mesenchymal transition (EMT) are other mechanisms of MDR. To zoom in on the correlation between altered glycosylation and drug resistance and to find out the putative biomarkers, MS-based glycoprotomics is a method of choice. Here, we report our N-glycoproteomics study of differential N-glycosylation from the whole cell lysate of MCF-7/ADR CSCs (adriamycin-resistant MCF-7 CSCs) relative to MCF-7 CSCs. Intact N-glycopeptides from both cells were enriched with ZIC-HILIC, isotopically diethylated, mixed with 1:1 ratio, and the mixture was analyzed by C18-RPLC-ESI-MS/MS (HCD with stepped NCE). Differentially expressed N-glycopeptides (DEGPs) were identified and quantified with database search using GPSeeker.

2023-07-04 | PXD036095 | Pride

N‑Glycoproteomics Study of Putative N‑Glycoprotein Biomarkers of Drug Resistance in MCF‑7/ADR Cells

Project description:Currently, drug resistance of anti-cancer therapy has become the main cause of low survival rate and poor prognosis. Full understanding of drug resistance mechanisms is an urgent request for further development of anti-cancer therapy and improvement of prognosis. Here we present our N-glycoproteomics study of putative N-glycoprotein biomarkers of drug resistance in doxorubicin resistance breast cancer cell line michigan cancer foundation-7 (MCF-7/ADR) relative to parental michigan cancer foundation-7 (MCF-7) cells. Intact N-glycopeptides (IDs) from MCF-7/ADR and MCF-7 cells were enriched with zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC), labeled with stable isotopic diethylation (SIDE), and analyzed with C18-RPLC-MS/MS (HCD with stepped normalized collision energies); these IDs were identifed with database search engine GPSeeker, and the diferentially expressed intact N-glycopeptides (DEGPs) were quantifed with GPSeekerQuan. With target-decoy searches and control of spectrum-level FDR≤1%, 322 intact N-glycopeptides were identifed; these intact N-glycopeptides come from the combination of 249 unique peptide backbones (corresponding to 234 intact N-glycoproteins) and 90 monosaccharide compositions (corresponding to 248 putative N-glycosites). The sequence structures of 165 IDs were confrmed with structure-diagnostic fragment ions. With the criteria of observation at least twice among the three technical replicates,≥1.5-fold change and p value<0.05, 20 DEGPs were quantifed, where fve of them were up-regulated and 15 of them were down-regulated; the corresponding intact N-glycoproteins as putative markers of drug resistance were discussed.

2023-12-30 | PXD036101 | Pride

Human stomach LC-MSMS China Human Proteome Project

Project description:With the optional setting of multiple stepped collisional energies (NCEs), higher-energy collisional dissociation (HCD) as available on Orbitrap instruments is a widely adopted dissociation method for intact N-glycopeptide characterization, where peptide backbones and N-glycan moieties are selectively fragmented at high and low NCEs, respectively. Initially, a dependent setting of a central value plus minus a variation is available to the users to set up NCEs, and the combination of 30±10% to give the energies 20%/30%/40% has been mostly adopted in the literature. With the recent availability of independent NCE setup, we found that the combination of 20%/30%/30% is better than 20%/30%/40%; in the analysis of complex intact N-glycopeptides enriched from gastric cancer tissues, total IDs with spectrum-level FDR≤1%, site-specific IDs with site-determining fragment ions and structure-specific IDs with structure-diagnostic fragment ions were increased by 42% (4,767->6,746), 57% (599->942), and 97% (1771->3495), respectively. This finding will benefit all the coming N-glycoproteomics studies using HCD as the dissociation method.

2020-05-11 | PXD016865 | Pride

Comparative glycomics study on the surface of SKOV3 versus IOSE80 cell lines

Project description:Both SKOV3 and IOSE80 cells were cultured and extracted the proteins. With sequential digestion by trypsin, enriched by Zwitterionic Hydrophilic Interaction chromatography (ZIC-HILIC), the intact N-glycopeptides were then isotopic diethyl labelled and analyzed using C18-RPLC-MS/MS (HCD).

2023-03-10 | PXD030927 | Pride

Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins using High-Resolution Mass Spectrometry

Project description:The global pandemic of severe acute pneumonia syndrome (COVID-19) caused by SARS-CoV-2 urgently calls for prevention and intervention strategies. The densely glycosylated spike (S) protein highly exposed on the viral surface is a determinant for virus binding and invasion into host cells as well as elicitation of a protective host immune response. Herein, we characterized the site-specific N-glycosylation of SARS-CoV-2 S protein using stepped collision energy (SCE) mass spectrometry (MS). Following digestion with two complementary proteases to cover all potential N-glycosylation sequons and integrated N-glycoproteomics analysis, we revealed the N-glycosylation profile of SARS-CoV-2 S proteins at the levels of intact N-glycopeptides and glycosites, along with the glycan composition and site-specific number of glycans. All 22 potential canonical N-glycosites were identified in S protein protomer. Of those, 18 N-glycosites were conserved between SARS-CoV and SARS-CoV-2 S proteins. Nearly all glycosites were preserved among the 753 SARS-CoV-2 genome sequences available in the public influenza database Global Initiative on Sharing All Influenza Data. By comparison, insect cell-expressed SARS-CoV-2 S protein contained 38 N-glycans, which were primarily assigned to the high-mannose type N-glycans, whereas the human cell-produced protein possessed up to 140 N-glycans largely belonging to the complex type. In particular, two N-glycosites located in the structurally exposed receptor-binding domain of S protein exhibited a relatively conserved N-glycan composition in human cells. This N-glycosylation profiling and determination of differences between distinct expression systems could shed light on the infection mechanism and promote development of vaccines and targeted drugs.

2020-10-05 | PXD018506 | Pride

Mucin-type O-glycosylation Landscapes of SARS-CoV-2 Spike Proteins

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

Hydrophilic interaction liquid chromatography contributes to integrated N-glycoproteomics analysis of human saliva for lung cancer

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

monoclonal antibody NIST8671 LC-MS/MS

Project description:The characteristics of monoclonal antibodies (mAbs) cohering various function effectors shows great expectation in therapy. Glycosylation, one of the common post-translational modifications, deeply influences the cohesion. It is necessary to grasp monosaccharide composition/sequence and glycan structures in mAbs. There have been comprehensive mass spectrometry characterization of N-glycosylation of mAbs, and monosaccharide compositions are deduced according to known biosynthetic rules. Our recently developed intact N-glycopeptide search engine GPSeeker has made unambiuous structure-specific characterization of N-glycosylation possible with structure-diagnostic fragment ions from selective fragmentattion of N-glycan moieties.

2022-04-14 | PXD032275 | Pride

EThcD-sceHCD-MS/MS-based Clinical N-glycoproteomics

Project description:Site-specific N-glycosylation characterization requires intact N-glycopeptide analysis based on suitable tandem mass spectrometry (MS/MS) method. Electron-transfer/higher-energy collisional dissociation (EThcD), stepped collision energy/higher-energy collisional dissociation (sceHCD), and higher-energy collision dissociation-product-dependent electron-transfer dissociation (HCD-pd-ETD) have emerged as valuable approaches for clinical glycoproteomics. However, each of them incurs some compromise, necessitating the performance comparisons when applied to the analysis of complex clinical samples (e.g., plasma, urine, cells, and tissue). Herein, we developed a hybrid mass spectrometry fragmentation method, EThcD-sceHCD, by combining EThcD and sceHCD. Moreover, we compared the performance of this method with those previous approaches (EThcD, sceHCD, HCD-pd-ETD, and sceHCD-pd-ETD) in the intact N-glycopeptide analysis, and determined its applicability for clinical N-glycoproteomic study.

2022-04-04 | PXD030288 | Pride

Sialo-glycoproteome of Human lung cancer cell by LC-MSMS

Project description:Alterations in protein glycosylation, especially the terminal sialylation, are closely correlated with physiological and pathological regulation. Due to the low ionization efficiency of sialo-glycopeptides and frequently observed dissociation of sialic acid residues during mass spectrometry analysis, tools to enrich the sialo-glycopeptides are essential to enhance detection sensitivity and identification efficiency in sialo-glycoproteomics. In this study, we present the first application of zwitterionic hydrophilic interaction chromatography (ZIC-cHILIC) material in StageTip for simultaneous enrichment and fractionation of intact glycopeptides at proteome scale. With the demonstrated enrichment specificity and identification depth, the stepwise-ZIC-cHILIC can be an efficient enrichment method for the discovery of glycosylation sites and native glycotope for many sample types.

2022-02-25 | PXD010456 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data