Computational protocol: The use of urinary and kidney SILAM proteomics to monitor kidney response to high dose morpholino oligonucleotides in the mdx mouse

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Protocol publication

[…] To measure urinary excretion of PMO we used a mass spectrometry approach for analysis of 10 μL urine aliquots collect at 0, 30 min, 45 min, 60 min, 90 min, 120 min, 180 min, and 360 min following injection of mdx-23 mice with single high dose PMO (800 mg/kg). Proteins were precipitated by addition of 200 μl methanol, followed by 1 h incubation at room temperature. Samples were then centrifuged for 10 min at 14,000 × g and supernatants containing the PMO were saved. The pellet was washed with 100 μl of 100 mM Tris, pH 7, then centrifuged again and supernatants pooled. Supernatants containing PMO were dried by vacuum centrifugation in Reacti-vials then resuspended in 50 μl of water, heated for 5 min at 60 °C and allowed to cool to room temperature. The sample was then processed using BioRad BioSpin 6 columns and Millipore C18 ZipTips using manufacturer's instructions. The ZipTip eluent was analyzed by static nanospray on a Thermo OrbitrapXL at 100,000 resolution and 800–2000 m/z. Spectra were deconvoluted using Thermo Xtract software. Signal intensities of injected morpholino were plotted vs. time. [...] For protein identification and quantification we used Integrated Proteomics Pipeline (IP2) version 1.42 software developed by Integrated Proteomics Applications, Inc. ( IP2 uses the Sequest search engine version 2010 (06 10 13 1836). Each raw MS and MS/MS file was searched against the forward and reverse UniProt mouse database (UniProt release 15.15, January 2013, 16,580 forward entries) tryptic peptides allowing two missed cleavages and the possible modifications of Met residue by oxidation (+15.99492 Da) and nitrogen atoms (+0.98 Da) to account for 15N labeled peptides. Because of the wide isotope distribution of 15N labeled peptides the mass tolerance was set at ±300 ppm (15 N) for MS and ±1.5 Da for MS/MS. Data were filtered by setting the protein false discovery rate (FDR) at 1%. Only proteins that were identified by at least two unique peptides were retained for further quantitative analysis. Census software (version 1.77), built into the IP2 platform, was used to determine the ratios of light to heavy peptide pairs using an extracted chromatogram approach. Quantitative data were filtered based on a determinant value of 0.5 and an outlier P-value of 0.1. […]

Pipeline specifications

Software tools xTract, Comet
Application MS-based untargeted proteomics
Organisms Mus musculus
Diseases Deficiency Diseases, Kidney Diseases, Muscular Dystrophy, Duchenne, Acute Kidney Injury
Chemicals Acetone, Methanol, Isoflurane, Morpholinos