Project PXD012985

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Biomedical Dataset
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Summary

Title

Translational regulation contributes to the secretory response of chondrocytic cells following exposure to Interleukin-1B

Description

Chondrocytes undergo changes to their protein translational capacity during osteoarthritis progression, but a study of how disease-relevant signals affect chondrocyte protein translation at the transcriptome level has not previously been performed. In this study we describe how the inflammatory cytokine IL-1B rapidly affects protein translation in the chondrosarcoma cell line SW1353. Using ribosome profiling we demonstrate that IL-1B induced altered translation of inflammatory-associated transcripts through differential translation and the use of multiple open reading frames. Proteomic analysis of the cellular layer and the conditioned media of these cells identified that proteins which were differentially translated were most readily detected in the secretome. We have produced combined ribosome profiling and proteomic datasets which provide a valuable resource in understanding the processes that are occuring during cytokine stimulation of chondrocytic cells.

Sample Processing Protocol

SW1353 were treated for 24 hours with and without IL-1B. Media was then harvested for secretome analysis and the cell layer was harvested for proteomic analysis. SW1353 were also treated for 3 hours with and without IL-1B. Cells were then harvested for ribosome profiling analysis.

Data Processing Protocol

For secretome analysis, media was collected and subjected to in-solution trypsin digestion and LC-MS/MS was performed using an Ultimate 3000 RSLC Nano system coupled to a QExtractive mass spectrophotometer. Label-free relative quantification software PEAKS was used to analyse RAW data files for identifications with Mascot. For the cell layer, protein lysates were digested with trypsin and mass spectrometry was performed using an Ultimate 3000 RSLC Nano system coupled to a QExtractive mass spectrometer. Proteins were considered significantly changed using a -10logP score >20 (equivalent to a P value of <0.01), a fold change >2 and at least 3 unique peptides. For ribosome profiling, samples were prepared accoriding to instructions from the ARTseq Ribosome Profiling Kit (Mammalian). Successfully amplified PCR libraries were quantified using a Qubit dsDNA High Sensitivity Kit and a Bioanalyser DNA High Sensitivity Kit. The size selected pooled library was assessed by bioanalyser and subsequently by qPCR using the Illumina Library Quantification Kit on a Roche Light Cycler LC480II. FASTQ files were uploaded to the RiboGalaxy server where adapter and ribosomeal RNA sequences were removed. riboSeqR was used via RiboGalaxy to align remaning reads to the human genome (GRCh38) before performing differential translation analysis.

Contact

Ben McDermott, University of Liverpool
Dr Simon Tew, Comparative Musculoskeletal Sciences Research Group Department of Musculoskeletal Biology Institute of Ageing and Chronic Disease University of Liverpool ( lab head )

Submission Date

05/03/2019

Publication Date

11/07/2019

Cell Type

fibroblast

Disease

disease free

Modification

Carbamidomethyl
Oxidation

Quantification

Peptide counting

Experiment Type

Shotgun proteomics

Assay count

12

Publication

Publication pending

Assay

Page 1 2
Page size 10
Showing 1 - 10 of 12 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 105608 sample1.mzid 1611 8666 6525 24031 0
2 105609 sample2.mzid 1609 9035 6647 24941 0
3 105610 sample3.mzid 3080 16505 6884 24590 0
4 105613 sample4.mzid 2830 14134 6029 23725 0
5 105614 sample5.mzid 1608 8268 6222 23255 0
6 105611 sample6.mzid 1642 9043 6723 25073 0
7 105612 sample1sec.mzid 686 6868 3047 17488 0
8 105606 sample2sec.mzid 552 4986 2261 14281 0
9 105617 sample3sec.mzid 728 7637 3242 18041 0
10 105607 sample4sec.mzid 53 331 161 1108 0