Dataset Information

Comparative Proteomic profiling of the rostral band of LAL muscle from SMA mice and litter mate controls

ABSTRACT: Mouse sample preparation: 'Severe' SMA mice (Smn-/-;SMN2+/+) and wild-type (Smn+/+;SMN2+/+) littermates at postnatal day 5 (P5) were sacrificed by chilling on ice and decapitation. Levator auris longus (LAL, from the back of the neck) muscles were dissected in oxygenated mammalian physiological saline, as previously described [PMID: 19640925]. LAL muscles were separated into rostral and caudal bands and quickly frozen on dry ice. The rostral band of LAL from each mouse was stored at -80C until sufficient tissue was collected for proteomics analysis. Proteomic analysis: Protein was extracted in MEBC Buffer (50 mM TRIS, 100 mM NaCl, 5 mM NaEDTA, 5 mM NaEGTA, 40 mM beta-glycerophosphate, 100 mM sodium fluoride (NaF), 100 mM sodium orthovanadate, 0.25% NP-40, 1 Roche 'complete' protease inhibitor tablet, pH 7.4). Protein concentration was determined by BCA P5 WT and KO Rostral). Then 10ug aliquots of each muscle type assay according to manufacturers instructions on solubilised muscle (were reduced with 10 mM DTT and alkylated with 50 mM iodoacetamide prior to digestion with trypsin (Roche, sequencing grade) overnight at 30C. Technical replicates (3 x 2.5 ug) of each digested muscle type were injected onto a nLCMS/ MS system (Ultimate 3000 (Dionex) coupled to a LTQ Orbitrap XL (Thermo Scientific). The peptides from each digest were separated over a 65 min linear gradient from 5-35% acetonitrile in 0.1% formic acid. The LTQ Orbitrap XL was configured with a TOP 5 methodology comprising a 60K resolution FT-MS full scan followed by IT-MS/MS scans for the 5 most intense peptide ions. The raw data was then imported into Progenesis LCMS for label free differential analysis and subsequent identification and quantification of relative ion abundance ratios, both up-regulated and down-regulated. Following alignment of MS data, principal component analysis and preliminary filtering (power >80%,P > 0.05), data was exported from Progenesis as a single mgf file per time point. These files were then used to identify individual peptide sequences using the Swiss-Prot database via Mascot Daemon (V2.4.0) due to the large file size. As an indication of identification certainty, the false discovery rate for peptide matches above identity threshold was 3.34% for P5.

INSTRUMENT(S): instrument model, LTQ Orbitrap XL

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Douglas Lamont

PROVIDER: PXD000488 | Pride | 2013-10-09

REPOSITORIES: Pride

ACCESS DATA

Publications

Label-free proteomics identifies Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers for spinal muscular atrophy.

Mutsaers Chantal A CA Lamont Douglas J DJ Hunter Gillian G Wishart Thomas M TM Gillingwater Thomas H TH

Genome medicine 20131018 10

<h4>Background</h4>Spinal muscular atrophy (SMA) is a neuromuscular disease resulting from mutations in the survival motor neuron 1 (SMN1) gene. Recent breakthroughs in preclinical research have highlighted several potential novel therapies for SMA, increasing the need for robust and sensitive clinical trial platforms for evaluating their effectiveness in human patient cohorts. Given that most clinical trials for SMA are likely to involve young children, there is a need for validated molecular b ...[more]

PMID: 24134804

Similar Datasets

Project description:Loss of ultrafiltration capacity in peritoneal dialysis (PD) patients is often associated with peritoneal vascular changes, manifest as diabetes-like vasculopathy and angiogenesis. The endothelial monolayer lining the vessel lumen controls vessel barrier function and thereby influences peritoneal substrate transport and ultrafiltration. In this study, we investigated the influence of exposure of human umbilical vein endothelial cells (HUVEC) to different PD fluids. HUVECs were exposed to experimental solutions for up to 24 h. All test ﬂuids were sterile-ﬁltered before usage. Each experiment consisted of three independent samples in biological replicates on separate culture plates. For PD fluid incubation, cells were first exposed to pure PD fluids (Dianeal PD4 3.86% glucose, Physioneal 3.86% glucose, Extraneal, all Baxter, Castlebar, Ireland), or to a lab-made GDP-free PD fluid or to normal medium without growth factors as control. Cells were subsequently exposed for 24 h to the above solutions diluted 1:1 with culture medium and brought to 2% FCS. Cells were then washed three times (250 mM sucrose, 10 mM Tris/HCl, pH 7) and lysed in 125 µL per well of lysis buffer (30 mM Tris, pH 8.5, 7 M urea, 2 M thiourea, 4% CHAPS, 1 mM EDTA, one tablet of Complete Protease Inhibitor (Roche, Basel; Switzerland) per 100 ml, and one tablet of PhosStop Protease Inhibitor (Roche) per 100 mL). Total protein concentration was determined with the Pierce 660 Kit (Thermo) per manufacturer’s manual. Lysates were stored at -80°C until further processing. We investigated the molecular mechanisms of endothelial cell pathology during in vitro PD fluid exposure by proteomic and bioinformatic analysis of the endothelial cell response to the PD fluid induced stress, integrating current understanding of PD-related cellular injury and stress responses (Bender et al., Nephrol Dial Transplant, 2011, doi:10.1093/ndt/gfq484; Kratochwill et al., BioMed research international, 2015, doi:10.1155/2015/628158; Kratochwill et al., J Proteome Res, 2009, doi:10.1021/pr800916s; Lechner et al., J Proteome Res, 2010, doi:10.1021/pr9011574). We then evaluated the effect of AlaGln addition to conventional PD fluid on these parameters in endothelial cells. This strategy allowed characterization of endothelial cell injury and stress responses during exposure to different PD fluid.

Dataset Information

Comparative Proteomic profiling of the rostral band of LAL muscle from SMA mice and litter mate controls

Publications

Label-free proteomics identifies Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers for spinal muscular atrophy.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure