Mass Spectrometry-Based Peptide Sequencing

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.