Urinary Proteome Analysis by Mass Spectrometry

A total of 44 urinary proteins resuspended in the buffer solution were digested overnight with Trypsin (Promega, United States) at 37°C temperature, acidified with TFA, redissolved with acetonitrile (ACN, Sigma, United States), eluted using two solvent buffer (A: 99.9% water and 0.1% formic acid; B: 79.9% ACN, 20% water, and 0.1% formic acid), and analyzed by using a Q Exactive HF quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to UltiMate 3,000 HPLC and UHPLC Systems (Thermo Fisher Scientific). Differential proteins were identified and quantified against the complete human proteins in the Uniprot database (2020.07.02) using Proteome Discover 2.4 software (Thermo Fisher Scientific) with SEQUEST and Mascot search engine (version 2.3.01, Matrix Science, London, United Kingdom). Then bioinformatics and statistical analysis were essentially performed as described previously: (1) differentially abundant proteins from discovery proteomics were selected using t-test after log2 transformed ratio based on the following criteria: p < 0.05 and FC < 0.83 or > 1.20; (2) the demographic data were presented as mean ± SEM, and statistical analysis was performed by the two-tailed t-test and one-way ANOVA for the comparison between groups; p < 0.05 was statistically significant. All procedure has been reported in details in our prior study (Chen et al., 2021 (link)).