Computational protocol: Proteomic Candidate Biomarkers of Drug-Induced Nephrotoxicity in the Rat

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Protocol publication

[…] Urine samples were analysed on a Dionex Ultimate 3000 RSLS nano flow system (Dionex, Camberly UK). After loading (5 µl) onto a Dionex 0.1×20 mm 5 µm C18 nano trap column at a flowrate of 5 µl/min in 0.1% formic acid and 2% acetonitrile, sample was eluted onto an Acclaim PepMap C18 nano column 75 µm×15 cm, 2 µm 100 Å at a flow rate of 0.3 µl/min. The trap and nano flow column were maintained at 35°C. The samples were eluted with a gradient of solvent A: 0.1% formic acid verses solvent B: 80% acetonitrile starting at 5% B rising to 50% B over 100 min.The eluant was ionized using a Proxeon nano spray ESI source (Thermo Fisher Hemel UK) operating in positive ion mode into an Orbitrap Velos FTMS. Ionization voltage was 2.5 kV and the capillary temperature was 200°C. The mass spectrometer was operated in MS/MS mode scanning from 380 to 2000 amu. The top 10 multiply charged ions were selected from each scan for MS/MS analysis using HCD at 35% collision energy. The resolution of ions in MS1 was 60,000 and 7,500 for HCD MS2. Data files were searched against the IPI rat non-redundant database using the Open Mass Spectrometry Search Algorithm (OMSSA, http://pubchem.ncbi.nlm.nih.gov/omssa) and SEQUEST (by using Thermo Proteome Discoverer), without any enzyme specificity. No fixed modification and oxidation of methionine and proline as variable modifications were selected. Mass error window of 10 ppm and 0.05 Da were allowed for MS and MS/MS, respectively. Peptide data were extracted using high peptide confidence and top one peptide rank filters. The OMSSA results were further optimized using COMPASS , 1% FDR was used as a cut-off value. The correlation between peptide charge at the working pH of 2 and CE-migration time was utilized to minimize false-positive identification rates : Calculated CE-migration time based on the number of basic amino acids was compared to the experimental migration time. Accepted were only those peptides which were found with both search algorithms (OMSSA and SEQUEST) and having a mass deviation below ±50 ppm and a CE-migration time deviation below ±2 min. […]

Pipeline specifications

Software tools OMSSA, Comet, Proteome Discoverer
Application MS-based untargeted proteomics
Organisms Rattus norvegicus
Diseases Kidney Diseases
Chemicals Cisplatin, Gentamicins