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Identification of enriched O-GlcNAc proteins in hypertrophic and normal mouse hearts

ABSTRACT: Bottom up proteomics of O-GlcNAc antibody enriched proteins from mouse heart tissue (hypertrophic vs. normal). O-GlcNAc proteins were labeled with a click chemistry kit with the tetramethylrhodamine (TAMRA) tag, then the labeled proteins were enriched with TAMRA antibody for proteomics experiments.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Aaron Olson

PROVIDER: MSV000088525 | MassIVE | Mon Dec 06 10:14:00 GMT 2021

SECONDARY ACCESSION(S): PXD030218

REPOSITORIES: MassIVE

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First comprehensive identification of cardiac proteins with putative increased O-GlcNAc levels during pressure overload hypertrophy.

Zhu Wei Zhong WZ Palazzo Teresa T Zhou Mowei M Ledee Dolena D Olson Heather M HM Paša-Tolić Ljiljana L Olson Aaron K AK

PloS one 20221026 10

Protein posttranslational modifications (PTMs) by O-GlcNAc globally rise during pressure-overload hypertrophy (POH). However, a major knowledge gap exists on the specific proteins undergoing changes in O-GlcNAc levels during POH primarily because this PTM is low abundance and easily lost during standard mass spectrometry (MS) conditions used for protein identification. Methodologies have emerged to enrich samples for O-GlcNAcylated proteins prior to MS analysis. Accordingly, our goal was to iden ...[more]

PMID: 36288343

Similar Datasets

Identification of enriched O-GlcNAc proteins in hypertrophic mouse hearts

Project description:We used C57/Bl6 mice subjected to sham or transverse aortic constriction (TAC) to create pressure-overload hypertrophy (POH). From the hearts, we stabilized and labelled the O-GlcNAc moiety with tetramethylrhodamine azide (TAMRA) before enriching by TAMRA immunoprecipitation (files with "TAMRAIP"). Global proteomics were performed on the non-enriched matching samples (files with "GlobalDigest"). Sham sample IDs: 529, 545, 547, 551, 552. TAC sample IDs: 524, 528, 562, 563, 564. O-GlcNAc proteins were labeled with a click chemistry kit with TAMRA tag (ThermoFisher Scientific), then the labeled proteins were enriched with TAMRA antibody for proteomics experiments. Data was searched against the UniProt mouse database using PEAKS Studio for label free quantitation. Protein abundances were log2 transformed, normalized to median, and missing values imputed using Perseus.

2022-02-07 | MSV000088797 | MassIVE

Genome-wide maps of O-GlcNAc-Oct4 binding regions in ZHBTc4 F-Oct4 mouse embryonic stem cells

Project description:We report that Oct4 is modified by O-GlcNAc in stem cells. To find O-GlcNAc-Oct4 target genes, we ChIPed Oct4 with Flag(Oct4) antibody and then O-GlcNAc modified proteins are enriched by sWGA beads (succinylated wheat germ agglutinin (sWGA), which specifically binds O-GlcNAc). Results show that several genes implicated in pluripotency regulation are bound by O-GlcNAc-Oct4 in their gene region.

2012-08-27 | GSE36388 | GEO

Genome-wide maps of O-GlcNAc-Oct4 binding regions in ZHBTc4 F-Oct4 mouse embryonic stem cells

2012-08-27 | E-GEOD-36388 | biostudies-arrayexpress

Genome-wide maps of GlcNAc bound proteins and Pho ChIP-seq in Drosophila S2 cells and pupae.

Project description:We developed a strategy to map GlcNAc modified proteins based on a chemical chromatin precipitation strategy. Drosophila S2 cells, as well as wild type (wt) and sxc null pupae, were fed with 100 uM Ac4GalNAz and the resulting DNA was cross-linked, sonicated, and purified. DNA strands cross-linked to GlcNAz proteins were isolated by congugating with alkyn-biotin by Staudinger ligation followed by streptavidin purification. The resulting DNA was constructed into libraries for sequencing. To asses the robustness of our strategy, we compared GalNAz ChIP-seq results in S2 cells with two other GlcNAc ChIP-seq strategies, using a mutant β-1,4-galactosyltransferase (GalT) and the lecting wheat germ agglutinin (WGA). Briefly, GalT was incubated with cross-linked, sonicated and purified DNA along with UDP-GalNAz. Ligation to biotin with click chemistry followed by streptavidin purification resulted in library ready material. For WGA and Pho ChIP-seq, cross-linked and purified DNA was incubated with pho antibody or WGA resin and purified followed by library preperation.

2017-07-18 | GSE84502 | GEO

Genome-wide maps of O-GlcNAc proteins in human breast cancer cells by next generation sequencing

Project description:We applied the chemical reporter-based metabolic labeling method to acquire O-GlcNAc modified proteins chromatin loci. Human breast cancer cell line MCF-7, as well as the genotoxic stress (Adriamycin) adapted cells MCF-7/ADR, were fed with 1 mM GalNAz. Metabolic labeled O-GlcNAz chromatin were crosslinked, sonicated and enriched by bioorthogonal chemistry. Then, the genomic DNA fragments bounded by O-GlcNAc mark were de-crosslinked, and constructed into libraries following by next-generation sequencing (Chemoselective O-GlcNAc chromatin sequencing, COGC-seq). To verify the robustness of this chemical reporter-based metabolic labeling method, we compared the results in MCF-7 and MCF-7/ADR cells with classical lectin succinylated wheat germ agglutinin (sWGA) ChIP-seq strategy. We also analyzed gene expression MCF-7 and MCF-7/ADR cells by RNA-seq.

2020-06-01 | GSE141698 | GEO

A chemical genetic method for monitoring genome-wide dynamics of O-GlcNAc turnover on chromatin-associated proteins.

Project description:O-linked N-acetylglucosamine (O-GlcNAc) is a necessary protein modification installed onto hundreds of nucleocytoplasmic proteins by O-GlcNAc transferase (OGT). Recently, we developed an antibody-free metabolic feeding approach that enables unbiased mapping of O-GlcNAcylated proteins in a genome-wide manner, providing insight into the regulation of genes by O-GlcNAcylated proteins within Drosophila. Here we apply this O-GlcNAc chemical mapping strategy to a time course (TC) feeding experiment within Drosophila larvae, generated the first ever TC ChIP-seq experiment performed on both a protein modification and within a living organism. TC metabolic labeling experiments were performed in wild-type and O-GlcNAc hydrolase (OGA) deficient Drosophila. Analysis of the resulting sequencing data revealed that a loss of OGA causes a global increase in the retention of O-GlcNAc modification on proteins bound to the genome, suggesting most nuclear proteins are sensitive to effects of O-GlcNAc cycling. Interestingly, some loci are more sensitive to the impacts of a loss of OGA compared to others. This study will present an improved understanding of the regulation of gene expression by O-GlcNAc while providing the broader community with experimental methods for time-resolved analysis of genome-wide binding by proteins.

2020-01-07 | GSE124785 | GEO

O-GlcNAc-modified proteins leaked from dead cells suppress fibrosis

Project description:Tissue fibrosis is induced by chronic inflammation accompanied by repeated injuries. Chronic inflammation alters fibroblasts and stellate cells, maintaining the tissues in myofibroblasts and activating stellate cells. This activation promotes the augmentation of these cells and the abundant production of extracellular matrices, such as collagen. Fibrotic tissue dysfunction is eventually induced by expelling parenchymal cells with these abundant collagen depositions. To recover from tissue fibrosis, the activation of myofibroblasts and stellate cells is suppressed by selectively targeting these cells. Here we show that O-N-acetylglucosamine (GlcNAc)-modified proteins leaked from dead cells and GlcNAc-bearing polymers mimicking the GlcNAc moiety of these proteins suppress the activation of these cells; administration of the GlcNAc-bearing polymer into carbon tetrachloride-treated mice can improve liver fibrosis. We found that a multivalent GlcNAc moiety structure elicited antifibrotic activities in myofibroblasts by interacting with cell surface vimentin and desmin. Therefore, since these cells highly express cytoskeletal proteins, vimentin and desmin, on the cell surface and have GlcNAc-binding activity, we suggest that O-GlcNAc-modified proteins leaked from dead cells can function as suppressors of fibrosis and hyperplasia in spontaneous recovery. Moreover, we anticipate that GlcNAc-bearing polymers mimicked by O-GlcNAc-modified proteins will be useful as new therapeutic tools for fibrosis and hyperplasia.

2022-03-30 | GSE199656 | GEO

Chromatin bound O-GlcNAc proteins related to stress response and transcriptional regulation.

Project description:O-GlcNAcylation performs a critical role in regulating stress response program and cellular homeostasis. However, systematic studies of O-GlcNAc regulated genotoxic stress-responsive transcriptional reprograming have been limited. The GalNAz metabolic labeled global chromatin-associated proteins were isolated from the cross-linked MCF-7 and MCF-7/ADR cells nucleus, and then modified by downstream click-chemistry. The de-crosslinked O-GlcNAz chromatin-bound proteins were enriched for label-free quantitative proteomics by LC-MS/MS.

2020-08-30 | PXD016713 |

Proteome-wide purification of O-GlcNAc proteins

Project description:Global O-GlcNAc profiling: proteome-wide purification and identification of O-GlcNAc proteins using GlcNAz metabolic labeling in combination with Click chemistry and label-free LC-MS/MS-based quantification. OGA inhibition: same workflow as 'Global O-GlcNAc profiling'; inhibition of O-GlcNAcase by a small molecule inhibitor. O-GlcNAc sites: identification of O-GlcNAc sites by metabolic labeling/Click chemistry followed by beta elimination Bioinformatics workflow of Global O-GlcNAc profiling and OGA inhibition experiments: Raw mass spectrometry files have been processed using Progenesis LC-MS (4.0) and exported as Mascot generic format for subsequent Mascot (2.3) database search. Mascot search results were processed using Mascot Percolator and the results have been imported back into Progenesis as well as imported to Scaffold (3.5.1). The Progenesis peptide quantification report has been exported as Excel file. Bioinformatics workflow of O-GlcNAc site identification: Raw mass spectrometry files have been processed using Mascot Distiller 2.3 and searched with Mascot. Mascot search results were processed using the Mascot Percolator and imported into Scaffold (3.5.1). After manual validation, peptide and protein identifications (Scaffold file) were imported into Scaffold PTM 2.0 for the estimation of site localization probabilities.

2013-01-10 | PXD000061 | Pride

O-GlcNAc proteome of invasive ductal breast carcinomas

Project description:O-GlcNAc proteins in LNM and non-LNM breast tumors of IDCs were identified by using the strategy of chemo-enzymatic labeling in combination with click chemistry and LC-MS/MS.

2015-09-22 | PXD002208 | Pride

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