Thermo proteome discoverer 1

LC-MS/MS data were processed initially uploading the raw spectra data into Thermo Proteome Discoverer 1.4 (Thermo Scientific, Hemel Hempstead, UK). Peak picking was performed under default settings for FTMS analysis such that only peptides with signal to noise ratio higher than 1.5 and belonging to precursor peptides between 700 and 8,000 Da were considered. Peptide and protein identification were performed with SEQUEST algorithm. An in house compiled database containing proteins from the latest version of the UniProt SwissProt database (2017) was compiled to include only Oncorhynchus mykiss. The search parameters were: Tryptic cleavage with 2 missed cleavages; static modification was carbamidomethyl of cysteines; allowed dynamic modifications were oxidation of methionine and phosphorlyation of serine, threonine, and tyrosine. Precursor tolerance was set at 10 ppm and MS2 tolerance was set at 0.05 Da. Resulting peptides and protein hits were further screened by excluding peptides with an error tolerance higher than 10 ppm and by accepting only those with an FDR<0.05. Protein identification was based on the presence of at least one unique peptide and quantification was based exclusively on unique peptide(s).

LC-MS/MS detection was performed on a hybrid quadrupole-time of flight LC-MS/MS mass spectrometer coupled with a nanospray source. Peptides were loaded onto a C18 trap column (Thermo Scientific, USA) and then eluted into a C18 analytical column (1.9 mm, 150 μm ×120mm, Thermo Scientific, USA). Three independent biological replicates were used for each sample, and peptides from these biological replicates were independently analyzed by the LC-MS/MS. Raw Thermo Scientific Q Exactive mass spectrometer was used to obtain the raw mass data in the RAW format. Thermo Proteome Discoverer 1.4 (Thermo Fisher, version 1.4.0.288) was used to convert the RAW file into the MGF format. ProteinPilot (software version: 4.5) was used to analyze the mass spectroscopy data. After exporting the data to Excel, DEPs were screened according to the criteria of standard false discovery rate (FDR) <0.01. Any proteins with a differential score >1.3, a peptide number ≥2, a difference >1.5 (upregulated) or <0.67 (downregulated), and a P < 0.05 were selected as a DAP. The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via PRIDE (16 (link)) partner repository with the dataset identifier PXD024100.

The iTRAQ labels for each group were as follows: 114-sham operation group, 116-NMT group, 118-MCAO model group, and 121-internal standard [10 (link)]. iTRAQ labeled samples were analyzed using a Gemini-NX 3u C18 column (250 x 4.6 mm, 5 μm). All experimental samples were monitored in real time [14 (link)]. A total of 20 fractions were collected, each of which used RPLC-MS analysis. Data were collected and processed using Thermo Fisher's Thermo Proteome Discoverer 1.4 (Version 1.4.0.288) software. AB Sciex's Protein Pilot TM Software 4.5 was used to process the raw acquired iTRAQ data files. The expression quantity, variation coefficient (CV), and p-values of each protein were analyzed on the basis of the screening criteria for DEPs, classed as a 1.2-fold change in expression (a multiple of the difference in expression of the same protein in both samples), CV ≤ 0.5, p-value < 0.05 for each protein.