Urine proteome changes in rats subcutaneously inoculated with approximately ten tumor cells.
Digested peptides (n = 27) were dissolved in 0.1% formic acid to a concentration of 0.5 µg/µL. For analysis, 1 µg of peptide from each sample was loaded into a trap column (75 µm × 2 cm, 3 µm, C18, 100 Å) at a flow rate of 0.25 µL/min and then separated with a reversed-phase analytical column (75 µm × 250 mm, 2 µm, C18, 100 Å). Peptides were eluted with a gradient extending from 4%–35% buffer B (0.1% formic acid in 80% acetonitrile) for 90 min and then analyzed with an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA). The MS data were acquired using the following parameters: i) data-dependent MS/MS scans per full scan were auquired at the top-speed mode; ii) MS scans had a resolution of 120,000, and MS/MS scans had a resolution of 30,000 in Orbitrap; iii) HCD collision energy was set to 30%; iv) dynamic exclusion was set to 30 s; v) the charge-state screening was set to +2 to +7; and vi) the maximum injection time was 45 ms. Each peptide sample was analyzed twice.
Raw data files (n=54) were searched using Mascot software (version 2.5.1, Matrix Science, London, UK) against the Swiss-Prot rat database (released in February 2017, containing 7,992 sequences). The parent ion tolerance was set to 10 ppm, and the fragment ion mass tolerance was set to 0.02 Da. The carbamidomethylation of cysteine was set as a fixed modification, and the oxidation of methionine was considered a variable modification. Two missed trypsin cleavage sites were allowed, and the specificity of trypsin digestion was set for cleavage after lysine or arginine. Dat files (n=54) were exported from Mascot software and then processed using Scaffold software (version 4.7.5, Proteome Software Inc., Portland, OR). The parameters were set as follows: both peptide and protein identifications were accepted at a false discovery rate (FDR) of less than 1.0% and proteins were identified with at least two unique peptides. Different samples were compared after normalization with the total spectra.
In this dataset,we present the mass spectrometry DAT files. Numbers 1-9 represent Rat 1-D0 to Rat 9- D0, numbers 10-18 represent Rat 1-D13 to Rat 9- D13 and numbers 19-27 represent Rat 1-D21 to Rat 9-D21.
