Computational protocol: Ageing-induced changes in the redox status of peripheral motor nerves imply an effect on redox signalling rather than oxidative damage

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Protocol publication

[…] Sciatic nerve samples from adult (n=4) and old (n=4) mice were prepared for proteomic analysis and quantification using a label-free quantitative proteomic approach that includes a differential cysteine labelling step as recently described . The approach allows simultaneous identification of up- and down regulated proteins between samples using global label free proteomics. In addition a targeted analysis of redox sensitive cysteine residues allows the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues within samples. The proteomics analysis was performed as previously described . The data-dependent label-free analysis was performed using an Ultimate 3000 RSLC™ nano system (Thermo Scientific, Hemel Hempstead, UK) coupled to a QExactive™ mass spectrometer (Thermo Scientific). The sample (5 µL corresponding to 250 ng of protein) was loaded onto the trapping column (Thermo Scientific, PepMap100, C18, 75 μm×20 mm), using partial loop injection, for 7 min at a flow rate of 4 μL/min with 0.1% (v/v) TFA. The sample was resolved on the analytical column (Easy-Spray C18 75 µm×500 mm×2 µm column) using a gradient of 97% A (0.1% formic acid) 3% B (99.9% ACN 0. 1% formic acid) to 60% A 40% B over 120 min at a flow rate of 300 nL/min. The programme used for data acquisition consisted of a 70,000 resolution full-scan MS scan (AGC set to 106 ions with a maximum fill time of 250 ms) the 10 most abundant peaks were selected for MS/MS using a 17,000 resolution scan (AGC set to 5×104 ions with a maximum fill time of 250 ms) with an ion selection window of 3 m/z and a normalized collision energy of 30. To avoid repeated selection of peptides for MS/MS the programme used a 30 s dynamic exclusion window.Raw spectra were converted to mascot generated files (.mgf) using Proteome Discoverer software (Thermo Scientific). The resulting mgf files were searched against the Uniprot Mouse database sequence database (12/05/2012, 16376 sequences) using an in-house Mascot server (Matrix Science, London, UK). Search parameters used were: peptide mass tolerances, 10 ppm; fragment mass tolerance, 0.01 Da, 1+, 2+ and 3+ ions; missed cleavages, 1; instrument type, ESI-TRAP. Variable modifications included were: d(0) N-ethylmaleimide (NEM), d(5) NEM, mono-, di- and tri-oxidation of cysteine residues and oxidation of methionine and a false discovery rate of<1%. Label-free relative quantification software PEAKS™ 7 (Bioinformatics Solutions Inc, Waterloo Canada) was used to analyse RAW data files against the same mouse protein database for identifications with Mascot . Proteins were considered significantly changed between adult and aged samples using a −10log P score>20 (equivalent to a P value<0.01) and using a quality value of >0.5. The full list of identified proteins including statistical analysis of protein and peptide features is included in . Cysteine containing peptides detected with identical amino acid sequences, and both d(0) and d(5) NEM modifications independently with an individual peptide ion Mascot score of>20 were considered redox peptides. Redox peptides detected from Proteome Discovery analyses of RAW files were selected for targeted analysis using m/z data and retention times with the open software Skyline™ . Targeted analysis applying m/z, retention times and fragmentation spectra for peptide selection allowed the estimation of the reduced:oxidized ratio (or d(0) / d(5) NEM) of the Cys residues using the individual parent ion intensities with Skyline™. The individual reduced:oxidized ratio for redox Cys peptides in each sample was used to calculate an average ratio of reduced:oxidized calculated for the specific cysteine residues. Intensities of targeted peptides are included in . […]

Pipeline specifications

Software tools Proteome Discoverer, Mascot Server
Application MS-based untargeted proteomics
Organisms Mus musculus
Chemicals Cysteine, Superoxides, Peroxynitrous Acid