Dataset Information

Proteomics Analysis of the Inferior Colliculus following Acute Hydrogen Sulfide

ABSTRACT: We used broad-spectrum proteomics to search for key molecules in H2S induced neurotoxicity. Mice were subjected to acute whole body exposure up to 750ppm of H2S at day1 for 40min, followed by 15min daily exposure. Following H2S exposure, inferior colliculi were harvested on day1, day2, and day4 and for proteomics analysis. Samples were processed according to a previously published method with some modifications, Meade et al. 2015. Briefly, inferior colliculus tissues were placed in 100 microliter of urea lysis buffer and homogenized with a handheld pestle homogenizer. Protein samples were further reduced and alkylated. Small aliquots of each sample were taken to measure protein concentration using the Bradford assay. The remaining samples were diluted and trypsin-digested overnight, followed by desalting using a C18 peptide trap from Michrom. The desalted samples were vacufuged prior to individual sample labeling using TMT6plex labels. Labeled samples of each exposure group were combined with the control for sample comparison. Peptides were separated on a Waters BEH C18 capillary column prior to online analysis using a 240min linear increasing gradient of acetonitrile with 0.1 percent formic acid. Following elution from the column, ions were generated using 2.6 kV on a taper tip in a New Objective nano source and entered into an LTQ Orbitrap Velos mass spectrometer. A full scan was taken in the LTQ, followed by data-dependent tandem mass spectrometry analysis of the top six peaks. MSMS analysis included collision-induced dissociation in the LTQ for structural information and higher energy collisional dissociation in the Orbitrap for quantitation. MSMS data were aligned and quantitated using MaxQuant 1.5.4.1 Analytics Platform with PTXQC quality control data management. Peptide alignment was executed with the mouse Uniprot protein database, enzyme trypsin; carboxymethyl, and oxidation, FDR 0.01 percent based on peptide q-value under standard settings.

INSTRUMENT(S): LTQ Orbitrap Velos

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

SUBMITTER: Andrea Hoffmann

PROVIDER: MSV000081650 | MassIVE | Thu Oct 26 08:39:00 BST 2017

SECONDARY ACCESSION(S): PXD008058

REPOSITORIES: MassIVE

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Publications

Broad spectrum proteomics analysis of the inferior colliculus following acute hydrogen sulfide exposure.

Kim Dong-Suk DS Anantharam Poojya P Hoffmann Andrea A Meade Mitchell L ML Grobe Nadja N Gearhart Jeffery M JM Whitley Elizabeth M EM Mahama Belinda B Rumbeiha Wilson K WK

Toxicology and applied pharmacology 20180619

Acute exposure to high concentrations of H2S causes severe brain injury and long-term neurological disorders, but the mechanisms involved are not known. To better understand the cellular and molecular mechanisms involved in acute H2S-induced neurodegeneration we used a broad-spectrum proteomic analysis approach to identify key molecules and molecular pathways involved in the pathogenesis of acute H2S-induced neurotoxicity and neurodegeneration. Mice were subjecte ...[more]

PMID: 29932956

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Project description:Isolated murine kidney glomeruli were lysed in buffer containing 8M urea, and proteins were reduced and alkylated. Proteins were digested with trypsin. After reverse-phase chomatrography, peptides were fractionated using strong cation exchange chromatography. Phosphopeptides were enriched using IMAC (FeNTA) columns. Peptides were analyzed by LC-MS/MS on a Q Exactive mass spectrometer or an LTQ Orbitrap XL mass spectrometer. Metadata for Orbitrap samples (MaxQuant Output files include „Orbi“) 1. General features – 1.1 Global descriptors a. Responsible person or role: Markus Rinschen. markus.rinschen@uk-koeln.de; tobias.lamkemeyer@uni-koeln.de b. Instrument manufacturer, model: LTQ-Orbitrap XL, Thermo Scientific c. customization 2. Ion sources – ESI fed by nLC 2 (Proxeon) 3. Post source component 3.1. Analyser: MS1 survey scan in an Orbitrap and MS2 analysed in Linear Trap. 3.2. Activation/Dissociation: CID 4. Spectrum and peak list generation and annotation 4.1. Data acquisition: MaxQuant v. 1.3.05 Top one method with a cycle of one full MS1 scan in the Orbitrap, followed by the fragmentation with dynamic exclusion window of 60 seconds and followed by the acquisition of 5 product ion scans generated in the LTQ analyser, and detected in the LTQ. Unselected fragmentation. For further details, see .raw files. URL of file: Filename 4.2. Data analysis: MaxQuant v 1.3.05 Parameters used in the generation of peak lists or processed spectra: see annotation in “parameters” file. The mouse uniprot reference database “complete proteome” obtained on January 2nd, 2013 was used. Metadata for QExactive samples (Max Quant output files include „QE“) 1. General features – 1.1 Global descriptors a. Responsible person or role: Markus Rinschen. markus.rinschen@uk-koeln.de; Marcus Krüger marcus.krueger@mpi-bn.mpg.de b. Instrument manufacturer, model: Q Exactive Thermo Scientific c. customization 2. Ion sources – ESI fed by nLC 1000 (Proxeon) 3. Post source component 3.1. Analyser: MS1 survey scan in an Orbitrap and MS2 analysed in Orbitrap/HCD cell 3.2. Activation/Dissociation: HCD 4. Spectrum and peak list generation and annotation 4.1. Data acquisition: MaxQuant v. 1.3.05 Top one method with a cycle of one full MS1 scan in the Orbitrap, followed by the fragmentation with dynamic exclusion window of 20 seconds in the HCD cell and followed by the acquisition of 10 product ion scans generated in the LTQ analyser, and detected in the LTQ. Unselected fragmentation. For further details, see .raw files. URL of file: Filename 4.2. Data analysis: MaxQuant v 1.3.05 Parameters used in the generation of peak lists or processed spectra: see annotation in “parameters” file. The mouse uniprot reference database “complete proteome” obtained on January 2nd, 2013 was used.

Dataset Information

Proteomics Analysis of the Inferior Colliculus following Acute Hydrogen Sulfide

Publications

Broad spectrum proteomics analysis of the inferior colliculus following acute hydrogen sulfide exposure.

Similar Datasets

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