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): LTQ Orbitrap XL

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Douglas Lamont

LAB HEAD: Douglas Lamont

PROVIDER: PXD000488 | Pride | 2013-10-09

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	230913P5WTvsKOrostralSwissProtMascotoutputforPRIDEF002355.dat	Other
	AdditionalMethodologyforPRIDE.docx	Other
	KO-R1.raw	Raw
	KO-R2.raw	Raw
	KO-R3.raw	Raw

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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

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