IP-MS experiment with biotin-iZMYND8-34 to analyze its associated proteins
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Jun Qin
PROVIDER: PXD054325 | iProX | Sun Jul 28 00:00:00 GMT+01:00 2024
REPOSITORIES: iProX
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Project description:The experiment was carried out to study the effect of loss of (p)ppGpp or cyclic di-GMP on the gene expression pattern of Mycobacterium smegmatis MC2 155 strains. The cells were cultured in MB7H9 broth containing 0.2% glycerol and 0.05% Tween 80. The transcriptome analysis was performed using GeneSpring GX12 software. Fold changes were calculated with respect to the median expression of wild type replicates. The number of differentially expressed genes (p <0.1) in rel knockout were 417 whereas those in dcpA knockout were 149. Earlier studies had shown that rel knockout and dcpA knockout had shown lower levels of glycopeptidolipids and polar lipids in the cell wall. The microarray data also corraborates our current findings. Organism : Mycobacterium smegmatis, Agilent Custom Mycobacterium smegmatis Gene Expression M.smegmatis_gxp_8X15K (AMADID: 029929) designed by Genotypic Technology Private Limited.
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Project description:Membranes of olfactory cilia were isolated from mouse olfactory epithelia. Sample fractionation was carried out in parallel by two methods: Proteins were fractionated by SDS-PAGE, the gel lanes were cut into bands and the proteins digested in-gel. Alternatively, proteins were digested in solution and peptides fractionated by strong cation exchange chromatography. Samples were analyzed using an LTQ-Orbitrap Velos. Mass spectrometric data of all replicates were searched together using the software MaxQuant (version 1.2.0.18) and its integrated search engine Andromeda against the UniProt reference proteome set for Mus musculus (http://www.uniprot.org/taxonomy/complete-proteomes; version of March 2, 2012, containing 54,231 entries) appended with a list of common contaminant proteins provided by MaxQuant. Database searches were performed with tryptic specificity allowing two missed cleavages and 7 ppm initial mass tolerance for precursor ions and 0.5 Da for fragment ions. Oxidation of methionine was considered as variable modification; fixed modifications were not included. Peptides and proteins were identified with a false discovery rate of 0.01.
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Project description:Triplicate chemostat cultivations were carried out with L. lactis subsp. lactis IL1403 at specific growth rates 0.1 and 0.5 h-1 on a chemically defined medium, where both, glucose and lysin (Lys) were jointly limiting the growth. After reaching the steady physiological state, medium was switched to heavy Lys (13C615N2-Lys) containing medium and incorporation of heavy amino acid into biomass was observed over the time course. Samples were collected before the medium switch (0 h) and 0.5; 1.5; 5; and 10 h after the medium switch. Cells were lysed in SDS lysis buffer and digested with trypsin according to Filter Aided Sample Preparation protocol (FASP). LC-MS/MS analysis were performed on Agilent 1200 series nanoflow system connected to a LTQ Orbitrap mass-spectrometer equipped with a nanoelectrospray ion source. Raw data files were analyzed with the MaxQuant software package (version 1.2.7.4). Generated peak lists were searched using the Andromeda search engine (built into MaxQuant) against L. lactis database (downloaded 28.06.2012 from http://www.genome.jp/kegg/genes.html). MaxQuant searches were performed with full tryptic specificity, a maximum of two missed cleavages and a mass tolerance of 0.5 Da for fragment ions. Carbamidomethylation of cysteine was set as a fixed modification and methionine oxidation and protein N-terminal acetylation were set as variable modification. The required false discovery rate (FDR) was set to 1% both for peptide and protein levels and the minimum required peptide length was set to six amino acids. In addition, "Match between runs" option with a time window of 1.5 min was allowed.
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2016-04-04 | PXD002344 | Pride
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