Proteome discover 2

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)).

GCF collected from healthy individuals and periodontitis patients was pooled and subjected to proteomic analysis. A total of 25 µg protein from the pooled GCF was used for proteomic analysis, and all sample preparation procedures for protein precipitation, peptide digestion, and fractionation were analyzed with a Q Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer. Peptide mass analysis was performed according to a previous method [12 (link)]. Data analysis was performed using Proteome Discover 2.4 software (Thermo Fisher, Waltham, MA, USA) for protein identification and label-free quantification. SEQUEST-HT, part of the Proteome Discoverer 2.4 software, was used for database searching against the UniProt human database. GCF proteomic analysis was conducted at the Proteomics Core Facility at the National Cancer Center (Goyang, Republic of Korea).

Sample processing for proteome analysis was performed according to the previous report [24 (link)]. Briefly, vitreous samples were digested with Trypsin/Lys-C Mix (Promega, #V5073). The resulting peptides were analyzed using UltiMate 3000 RSLCnano-flow HPLC (Thermo Fisher Scientific, Waltham, MA, USA) equipped with 0.075 mm × 250 mm AURORA column (IonOpticks, Melbourne, Australia) combined with Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific). From the datasets obtained from LC-MS/MS, the proteins were identified by searching the SwissProt Human Database using the Mascot (Matrix Science, London, UK) or Sequest HT (Thermo Fisher Scientific) search engine. Protein identification and quantification was performed using the Proteome Discover 2.4 software (Thermo Fisher Science).