Thermo ultimate 3000 uhplc

The S180 cells were treated with 5 μM MS275 or 0.4% (v/v) DMSO as control group for 48-h incubation and the sufficient samples were submitted for iTRAQ quantitative proteomics analysis (BGI, Shenzhen, China). Protein extraction, SDS-PAGE purification, protein digestion and peptide quantification was dealt as reference [1 (link)]. The peptide samples were respectively labeled using the iTRAQ Reagent-4plex Multiplex Kit (AB SCIEX) according to the manufacturer's instructions. The labeled peptide fractionation was carried out by using Shimadzu LC-20 AB liquid phase system with 5  μm 4.6 × 250 mm Gemini C18 column and followed by HPLC (Thermo UltiMate 3000 UHPLC). The nanoliter liquid phase separation end was directly connected to the mass spectrometer with a tandem mass spectrometer Q-Exactive HF X (Thermo Fisher Scientific, San Jose, CA). The MS/MS data were searched against the Mascot database (uniprot-human 20151227.fasta) for peptide identification and quantification. The searching result of peptides was filtered by FDR p value with a cut off of 0.05. Based on statistical dispersion of the dataset, ratio of > 1.5 or < 0.667 was used as a strict significance cutoff to acquire a short list of the differentially distributed proteins as indicated in the data legends.

The dried peptide samples were reconstituted with mobile phase A (2% ACN, 0.1% FA), centrifuged at 20,000 g for 10 min, and the supernatant was taken for injection. Separation was performed on a Thermo UltiMate 3000 UHPLC (Thermo Fisher Scientific).

iTRAQ proteomics was applied to analyze synovial tissue of the knee joint from the normal group, model group, and T1 group. The protein extraction, quality control, and proteolysis were performed according to the protocol. The iTRAQ® reagent 8 plex (AB Sciex Pte. Ltd., USA) was used to label peptides. The Shimadzu LC-20AB liquid phase system (Shimadzu, Japan) was used for peptide fractionation, and peptide samples were separated by Thermo UltiMate 3000 UHPLC (Thermo Fisher Scientific, San Jose, USA). The peptides separated by liquid phase chromatography were ionized and then passed to a tandem mass spectrometer Q-Exactive HF X (Thermo Fisher Scientific, San Jose, CA, USA) for data-dependent acquisition (DDA) mode detection.
The raw mass spectrometry (MS) data was converted into Mascot Generic Format (MGF) format, which was then searched by the local Mascot server. In addition, quality control was performed, and IQuant soft was applied to the quantification of proteins [26 (link)]. To assess the confidence of peptides, the Professional Scrum Master™ levels (PSMs) were prefiltered at a PSM-level false discovery rate (FDR) of 1%. The protein FDR was also set at 1% (protein − level FDR ≤ 0.01).