For each sample, thirty-five µg of proteins were subjected to reduction, alkylation, digestion and labelling using 6-plex Tandem Mass Tag reagents, according to manufacturer instructions (Thermo Scientific, New York, NY, USA) as described previously [18 (link),19 (link)]. A pool with 35 µg protein from each sample was included as internal standard and data from each sample and protein was calculated as a ratio of the internal standard.
The liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed using Dionex Ultimate 3000 RSLC nano-flow system (Dionex, Camberley, UK) and Orbitrap Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) as described elsewhere [58 (link)]. For peptide identification and relative quantification, SEQUEST algorithm, Proteome Discoverer (version 2.0., Thermo Fisher Scientific), was used. NCBI database search against Canis Lupus FASTA files was performed considering two trypsin missed cleavage sites, precursor tolerance of 10 ppm and fragment mass tolerance of 0.02 Da. The false discovery rate (FDR) for peptide identification was set at 1% and Percolator algorithm within the Proteome Discoverer workflow was used.
The liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed using Dionex Ultimate 3000 RSLC nano-flow system (Dionex, Camberley, UK) and Orbitrap Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) as described elsewhere [58 (link)]. For peptide identification and relative quantification, SEQUEST algorithm, Proteome Discoverer (version 2.0., Thermo Fisher Scientific), was used. NCBI database search against Canis Lupus FASTA files was performed considering two trypsin missed cleavage sites, precursor tolerance of 10 ppm and fragment mass tolerance of 0.02 Da. The false discovery rate (FDR) for peptide identification was set at 1% and Percolator algorithm within the Proteome Discoverer workflow was used.
Free full text: Click here
