Dionex ultimate 3000 rslc nano flow system

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.

For sequencing of peptides the urine samples were analysed on a Dionex Ultimate 3000 RSLC nano flow system (Dionex, Camberly, UK) coupled to an Orbitrap Velos MS instrument (Thermo Fisher Scientific) as described in [38 (link)]. Data files were analysed using Proteome Discoverer 1.2 (activation type: HCD; min-max precursor mass: 790-6,000; precursor mass tolerance: 10 ppm; fragment mass tolerance: 0.05 Da; S/N threshold: 1) and were searched against the Uniprot human non-redundant database without enzyme specificity. No fixed modifications were selected, oxidation of methionine, lysine and proline were selected as variable modifications. The peptide data were extracted using high confidence peptides, defined by an Xcorr ≥ 1.9, a delta mass between experimental and theoretical mass ± 5 ppm, absence of cysteines in the sequence (since cysteines without reduction and alkylation form disulphide bonds), absence of oxidised proline in protein precursors other than collagens or elastin, and top one peptide rank filters. For further validation of obtained peptide identifications, the strict correlation between peptide charge at the working pH of 2 and CE-migration time was used to prevent false identifications [39 (link)]. Only the sequenced peptides were further considered.

From each sample, 35 μg of protein was subjected to reduction, alkylation, digestion, and labeling using 6-plex TMT reagents according to manufacturer instructions (Thermo Fisher Scientific, Waltham, MA USA) as described previously (23 (link)).
Dionex Ultimate 3000 RSLC nano-flow system (Dionex, Camberley, UK) and Orbitrap Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) were used for the liquid chromatography–tandem mass spectrometry, as described elsewhere (23 (link)). SEQUEST algorithm, Proteome Discoverer (version 2.0., Thermo Fisher Scientific), was used for peptide identification and relative quantification. 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. Percolator algorithm within the Proteome Discoverer workflow was used to determine the false discovery rate (FDR) for peptide identification, which was set at 1%. In cases where GI accession number was described as unnamed, BLAST analysis and sequence comparison public database (NCBI) information were performed, and the result with the higher identity and query cover were included (>98% in all cases).