Dataset Information

Proteome-wide purification of O-GlcNAc proteins

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap, LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hannes Hahne

PROVIDER: PXD000061 | Pride | 2013-01-10

REPOSITORIES: Pride

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Publications

Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry.

Hahne Hannes H Sobotzki Nadine N Nyberg Tamara T Helm Dominic D Borodkin Vladimir S VS van Aalten Daan M F DM Agnew Brian B Kuster Bernhard B

Journal of proteome research 20130118 2

The post-translational modification of proteins with N-acetylglucosamine (O-GlcNAc) is involved in the regulation of a wide variety of cellular processes and associated with a number of chronic diseases. Despite its emerging biological significance, the systematic identification of O-GlcNAc proteins is still challenging. In the present study, we demonstrate a significantly improved O-GlcNAc protein enrichment procedure, which exploits metabolic labeling of cells by azide-modified GlcNAc and copp ...[more]

PMID: 23301498

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Project description:Drosophila development is a complex and dynamic process regulated, in part, by members of the Polycomb (Pc), Trithorax (Trx) and Compass chromatin modifier complexes. O-GlcNAc Transferase (OGT/SXC) is essential for Pc repression suggesting that the O-GlcNAcylation of proteins plays a key role in regulating development. OGT transfers N-acetyl-D-glucosamine (GlcNAc) onto hydroxyl groups of serine or threonine residues of key transcriptional regulators using the nutrient-derived UDP-GlcNAc as a substrate, which is dynamically removed by O-GlcNAcase (OGA). We performed ChIP-chip and microarray analysis after OGT or OGA RNAi knockdown in Drosophila S2 cells and found that O-GlcNAc was elevated genome wide particularly at genes related to mitosis and cell cycle in OGA RNAi cells, but not at sites co-occupied by Pc member Pleiohomeotic (Pho), such as the Hox and NK homeobox gene clusters. Microarray analysis suggested that altered O-GlcNAc cycling perturbed the expression of genes associated with morphogenesis and cell cycle regulation. To examine the in vivo consequences of disturbed O-GlcNAc cycling in the whole animal, we produced a null allele of oga (ogadel.1) in Drosophila. Epigenetic activators including Trx group members Trithorax (Trx), Absent small or homeotic discs 1 (Ash1) and Compass member Set1 histone methyltransferases are O-GlcNAc modified in ogadel.1 mutants. ogadel.1 mutants displayed altered expression of a distinct set of cell cycle related genes in ovaries. Our results suggest that the loss of OGA could affect epigenetic machinery by accumulating O-GlcNAc on numerous chromatin factors including Trx, Ash1 and Set1 in Drosophila. We performed affymetrix tilingarray analysis after OGT or OGA RNAi knockdown in Drosophila S2 cells to find if that O-GlcNAc was elevated genome wide particularly at genes related to mitosis and cell cycle in OGA RNAi cells, but not at sites co-occupied by Pc member Pleiohomeotic (Pho), ------------------------------- This represents the gene expression component only

Project description:The bacterium Wolbachia (order Rickettsiales), representing perhaps the most abundant vertically transmitted microbe worldwide, infects arthropods and filarial nematodes. In arthropods, Wolbachia can induce reproductive alterations and interfere with the transmission of several arthropod-borne pathogens. Additionally, Wolbachia is an obligate mutualist of the filarial parasites that cause lymphatic filariasis and onchocerciasis in the tropics. Targeting Wolbachia with tetracycline antibiotics leads to sterilisation and ultimately death of adult filariae. However, several weeks of treatment are required, restricting the implementation of this control strategy. To date, the response of Wolbachia to stress has not been investigated and almost nothing is known about global regulation of gene expression in this organism. We exposed an arthropod Wolbachia strain to doxycycline in vitro, and analysed differential expression by directional RNA-seq and label-free, quantitative proteomics. For proteomic analysis, Wolbachia-enriched material was purified further using additional filtration and Percoll gradients. Proteins were solubilised by sonication in a MS safe detergent, reduced with dithiothreitol and alkylated with iodoacetamide. Proteomic grade trypsin was added at a protein:trypsin ratio of 50:1, and samples were incubated at 37 C overnight prior to removal of detergent by trifluoroacetic acid (TFA) precipitation. Peptide mixtures were analysed by nano LC-MSMS using a LTQ-Orbitrap Velos (ThermoFisher Scientific). In one control sample, peptides were fractionated by strong anion exchange (SAX) using commercially available SAX spin filters (Thermo Scientific). Data was imported into Progenesis LC-MS (Nonlinear Dynamics) and runs were time-aligned using default settings. Peaks were picked by the software. Peptide intensities were normalised against an auto-selected reference run and differences in protein expression and associated ANOVA P-values between seven control and nine doxycycline-treated biological replicates were calculated by Progenesis LC-MS. Spectral data were transformed to MGF files with Progenesis LC-MS and exported for peptide identification using the Mascot search engine (Matrix Science). Search parameters were set to a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.5 Da, while one missed tryptic cleavage was permitted. Carbamidomethylation (cysteine) was set as a fixed modification and oxidation (methionine) set as a variable modification. Mascot search results were further processed using the machine learning algorithm Percolator. The false discovery rate was less than 1%, and individual ion scores greater than 13 were considered to indicate identity or extensive homology (P less than 0.05). Data were imported into Progenesis LC-MS as XML files. For a protein to be considered differentially expressed, a FC of greater than 1.5 and a P-value of less than 0.01 was required, supported by greater than 2 unique peptides.

Dataset Information

Proteome-wide purification of O-GlcNAc proteins

Publications

Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry.

Similar Datasets

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