Q exactive hf quadrupole orbitrap mass spectrometer

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)).

Lyophilized histone peptides were resuspended in 0.1% formic acid (FA) and analyzed on a Dionex U3000 ultra performance liquid chromatography system coupled to a Q-Exactive HF quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific). A Waters BEH 300Å C18 reversed phase capillary column (150 mm × 75 μm, 1.7 μm) was used for separation. Water with 0.1% FA and acetonitrile with 0.1% FA were used as mobile phases A and B, respectively. The flow rate was set to 0.300 μl/min. About 2 μl of peptide sample was injected onto the column and separated over a 120-min gradient as follows: 0–1 min 3–10% B; 1–90 min 10–35% B; 90–92 min 35–95% B; 92–102 min 95% B; 102–105 min 95–3% B; 105–120 min 3% B. The data were acquired under data dependent acquisition mode (DDA, top 20). Mass spectrometric conditions were as follows: spray voltage of 2.8 kV, no sheath and auxiliary gas flow; heated capillary temperature of 275°C, normalized high-energy collision dissociation (HCD) collision energy of 33%, resolution of 120 000 for full scan, resolution of 60 000 for MS/MS scan, automatic gain control of 2e5, maximum ion injection time of 100 ms, isolation window of 1.6 and fixed first mass of 110 m/z.

HCT116 and HCTR cells were seeded at a density of 0.25 × 10⁶ cells/mL in 1 mL medium in 12-well plates and left to recover overnight. The medium was replaced and the cells were treated with 100 µM BOLD-100 or the corresponding concentration of DMSO for 24 h. The cells were washed 3 times with PBS (pH 7.4), quenched with liquid nitrogen, and stored at −80 °C until further processing. The metabolomics experiments were carried out as described before [31 (link)]. In short, liquid chromatography high-resolution mass spectrometry (LC-MS) measurement with a Thermo Scientific Q Exactive HF quadrupole-Orbitrap mass spectrometer was utilized in full mass scan mode (both positive and negative ionization-mode) at a resolution of 120,000. External calibration with fully labelled ¹³C internal standards ISOtopic solutions (Vienna, Austria) was used for quantification. The obtained absolute metabolite amounts (pmol) were normalized to the total protein content in their respective well (µg) with the Thermo BCA kit, according to manufacturer’s instructions.