Itraq reagent 8 plex multiplex kit

Proteins were precipitated following the addition of a fourfold volume of cold acetone containing 10 mM DTT for about 2 h at -20°C, and centrifugation at 12,000 × g for 20 min at 4°C, followed by air-drying and dissolution with 100 μL TEAB dissolution buffer. The Bradford method was used to quantify each protein sample. From each sample, 100 μg of protein was reduced, alkylated, and then digested by Trypsin (Promega, Madison, WI, USA) at 37°C for 16 h, with the ratio of protein : Trypsin = 20:1, and labeled using the iTRAQ Reagent-8 plex Multiplex Kit (SCIEX, Framingham, MA, USA) according to the manufacturer’s protocol. Two biological replicates were carried out for the control group, and three biological replicates for the FFA and HH groups. The control group samples were labeled with 113 and 114; FFA group samples were labeled with 115, 116, and 117; HH group samples were labeled with 118, 119, and 121.

The protein contents in the mice serum were evaluated by the liquid chromatography in combination with tandem mass spectroscopy (LC-MS/MS), which was performed by Shanghai Genechem Co., Ltd. Briefly, protein digestion and labelling with iTRAQ Reagent 8-plex Multiplex kit (Sciex) were performed according to manufacturer's instruction. After centrifugation (12,000 g, 4 °C, 10 min) to remove the remaining debris, the supernatant was collected and the protein concentration was determined with a BCA kit (Q10) according to the manufacturer's instruction. The tryptic peptides were then digested into fractions and separated by high pH reverse-phase HPLC based on Agilent 300 Extend C18 column (5 mm particles, 4.6 mm ID, 250 mm length) and subjected to a NSI source followed by tandem mass spectrometry (MS/MS) in a Q ExactiveTM Plus (Thermo, MA, United States) coupled online UPLC. The MS/MS data were processed with the Maxquant search engine (v.1.5.2.8).

The differentially expressed proteins in rat bladder tissues were comprehensively characterized by the iTRAQ (isobaric tags for relative and absolute quantitation) as previously reported with minor modifications (Gao et al., 2020 (link)). Briefly, total protein samples extracted from rat bladder tissues via acetone precipitation were quantitated by the Bradford method. About 300 ug rat bladder proteins from each group were mixed with 30 ul SDT buffer, boiled at 100°C for 5 min, mixed with 200 ul UA buffer (8 M Urea, and 150 mM TrisHCl pH8.0), and incubated with 50 mM IAA for 30 min in darkness. Subsequently, rat bladder proteins were digested by overnight incubation with 40 µl Trypsin solution (4 µg Trypsin dissolved in 40 µl Dissolution buffer) at 37°C for 17–18 h. After centrifuge at 14000 g for 10 min, the collected peptides were quantitated by measuring the OD280 values. Approximately 100 ug rat bladder peptides in each group were subjected to labeling with the iTRAQ Reagent-8 plex Multiplex Kit (AB SCIEX) following the manufacturer’s instructions.

Firstly, total proteins were extracted from mouse lungs tissue and filtered by 10 KDa ultrafiltration tube, and the obtained penetrating fluid was the peptide sample, to which we added 1% trifluoroacetic acid (TFA) to adjust the pH to 2–3. Then, 100 μg peptide sample was taken for enzyming and desalting and was dried with a vacuum concentrator. We dissolved the peptide samples in 20 μL of dissolution buffer (0.5 M tetraethylammonium bromide, TEAB), added 70 μL of isopropanol, and centrifuged it with shaking. Samples were labeled with iTRAQ Reagent-8 plex Multiplex Kit (AB Sciex, MA, USA) according to the manufacturer’s instructions. Then, the peptide samples were desalted and dried with a vacuum concentrator. Peptide samples were diluted into 1 μg/μL on-board buffer and the volume was set to 8 μL, and they were analyzed using Triple TOF 5600 + LC/MS system (AB Sciex, MA, USA) for mass spectrometry data acquisition. Proteopilot 4.5 software (July 2012; ab SCIEX) was used to retrieve and analyze the mass-spectrometer data. To be considered as being differentially expressed, peptides were required to have a p ≤ 0.05 and fold change ≥1.5 (defined as up-regulated) or fold change ≤0.67 (defined as down-regulated). General properties of the DEPs were analyzed by Volcano maps, heat maps, GO, and KEGG bioinformatics analysis.

For each sample, 100 μg of protein was used and applied to the following process. The disulfide bond of the supernatant was reduced with 25 mM DTT at 60 °C for 1 h, cysteine was blocked with 55 mM iodacetamide in the dark for 45 min, and then the mixture was added to Millipore Amicon Ultra-15 (MWCO 10 k), followed by washing three times using iTRAQ Dissolution Buffer (AB Sciex, USA) and the addition of 4 μg of Trypsin Gold (Promega, USA) for protein digestion overnight at 37 °C. The resulting peptide solution was collected after centrifugation at 12,000 g for 20 min. The peptide solution was then labeled using the iTRAQ Reagent-8plex Multiplex Kit (AB Sciex, USA) according to the manufacturer’s instructions. Three biological replicates for two treatments were applied to the iTRAQ reagent (6-plex) labeling as follows: 24 h-low-1 (115), 24 h-low-2 (116), 24 h-low-3 (117), 24 h-high-1(118), 24 h-high-2 (119), and 24 h-high-3 (121). MALDI-TOF-TOF (AB Sciex 4800 Plus) was applied to check the labeling efficiency and quantitative accuracy.