Triple tof 5600 system

Samples were subjected to untargeted LC-MS measurements using a TripleTOF 5600+ system (Sciex, Framingham, MA, USA) and MarkerView software (version 1.2.1, Sciex) [24 (link)]. The peaks table consisted of one normalized peak area column per sample, as well as a mass value (m/z, mass-to-charge ratio) and retention time (min) column common to all samples. The nonzero peak areas were converted to common logarithms. LMIs with good discriminative ability (e.g., distinguishing MRI (+)/cytology (+) from MRI (−)/cytology (−) groups) were identified using the logarithmic peak area. The procedures for assessing individual LMIs were as follows. (1) For each LMI, a discrimination threshold value was determined (using increments of 0.01), wherein the sum of the sensitivity and specificity was highest (if adjacent threshold values had the same highest discrimination performance, the mean value was used). (2) Individual LMIs showing good discrimination (a summed sensitivity and specificity of 160% or higher) were set aside for subsequent analysis. The same procedures as above were applied to discover individual LMI candidates for distinguishing MRI (+)/cytology (−) or MRI (−)/cytology (+) from MRI (−)/cytology (−) groups, after replacing the MRI (+)/cytology (+) group with the MRI (+)/cytology (−) group or MRI (−)/cytology (+) group.

Each fraction was resuspended in buffer A (5% ACN and 0.1% FA) and centrifuged for 10 min; the average final peptide concentration was 0.5 μg/μL. The supernatant was loaded onto a 2 cm C18 trap column on a LC-20AD nano-HPLC system (Shimadzu, Kyoto, Japan). The peptides were then eluted on a 10 cm analytical C18 column (inner diameter, 75 μm). The samples were loaded as follows: 8 μL/min for 4 min; 35-min gradient running at 300 nL/min; a 5-min linear gradient to 60%; a 2 min linear gradient to 80%; 80% B for 4 min and 5% for 1 min. Data acquisition was performed using a TripleTOF^® 5600 system (Sciex, Concord, ON, Canada) that was fitted with a NanoSpray^® III source and a pulled quartz tip as an emitter^{75 (link)}.

Equal concentrations of protein samples were trypsinized, and digested proteins were analyzed using a TripleTOF 5600 MS (AB Sciex, Foster City, CA, United States) equipped with an Eksigent MicroLC 200 system (Eksigent, Dublin, CA, United States) with an Eksigent C18 reverse-phase column (150 × 0.3 mm, 3 μm, 120 Å) (Sharma et al., 2019a (link)). For protein identification, spectral libraries were generated using information-dependent acquisition (IDA) mode after injecting 2 gm of tryptic digest on the column using an Eksigent NanoLC-Ultra^TM 2D Plus system coupled with a SCIEX Triple TOF^® 5600 system fitted with a NanoSpray III source. The samples were loaded on the trap (Eksigent Chrom XP 350 μm × 0.5 mm, 3 μm, 120 Å) and washed for 30 min at 3 μl/min. A 120 min gradient in multiple steps (ranging from 5 to 50% acetonitrile in water containing 0.1% formic acid) was set up to elute the peptides from the ChromXP 3-C18 (0.075 × 150 mm, 3 μm, 120 Å) analytical column. Technical replicates of the nanoLC-TripleTOF 5600 MS experiments were performed.

Data acquisition was performed using a TripleTOF 5600 System (SCIEX, Framingham, MA, USA) fitted with a Nanospray III source (SCIEX, Framingham, MA, USA) and a pulled quartz tip as the emitter (New Objectives, Woburn, MA, USA). The high-sensitivity mode was set for survey scans. Raw data files were transformed into MGF files using ProteoWizard tool and the exported MGF files were searched by Mascot 2.3.02 (Matrix Science, Boston, MA, USA). NCBInr and Swiss Prot/UniProt database searches were performed for protein identification. In addition, the transcriptome database was useful for protein identification. The automated IQuant software quantitatively analyzed the labeled peptides with isobaric tags [82 (link)]. We used the ratio of the standard deviation sigma to the mean (CV) to evaluate the reproducibility. The lower the CV value, the better the reproducibility. In this project, we also set S1/S0, S2/S1 and S3/S2 as comparison groups. p values, representing the probability that the protein is differentially expressed, of less than 0.05 and fold change ≥ 1.2 were set as the significant thresholds for differential expression. Then, we searched against the GO, COG and KEGG databases to classify and identify differentially expressed proteins (DEPs). Additionally, classification and functional enrichments for DEPs by GO and KEGG pathways were performed (p ≤ 0.05).

The iTRAQ labeling method was applied to investigate the proteome changes after quercetin treatment for 12 h according to manufacturer’s instructions and as described previously (Wang et al., 2016 (link)). Briefly, after protein digestion, the peptides were labeled with four respective isobaric tags for 2 h and then pooled together. The contaminants were removed by an iTRAQ Method Development Kit (SCIEX, 4352160) using the strong cation exchange chromatography technique. Dried samples were reconstituted with diluent of 2% acetonitrile and 0.05% formic acid. After using an Eksigent NanoLCUltra system coupled to the cHiPLCNanoflex system (Eksigent, United States), the iTRAQ labeled peptides were detected by MS/MS with a TripleTOF 5600 system (SCIEX) set and identified by the Paragon algorithm with Protein Pilot TM Software 4.5 (SCIEX).

The 1D and 2D NMR spectra were recorded using an ASCEND 600 spectrometer (Bruker BioSpin Gmbh, Rheinstetten, Germany) at 298 K. All chemical shifts are reported in ppm from tetramethylsilane, using solvent resonances resulting from incomplete deuteration as the internal reference. The HR-ESIMS data were obtained using a TripleTOF 5600+ system (SCIEX, Framingham, MA, USA). Specific optical rotations were measured using an Autopol III S2 Polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). IR spectra were recorded using an FT/IR-4100 spectrometer (JASCO Inc., Easton, MD, USA), while the UV–visible spectra were measured using a Shimadzu UV-1650PC spectrophotometer. Semi-preparative HPLC was performed using a Waters^TM 1525 binary pump and a UV/Visible 2489 detector (Tepnel Pharma Services Limited, Livingston, Scotland). HPLC was performed using a YMC-Pack Pro C-18 column (250 × 10 mm l.D., S-5 µm, 12 nm). Silica gel (0.04–0.063 mm particle size, Merck) and RP-18 (0.04–0.063 mm particle size, Merck) were used for carrying out flash column chromatography. Thin layer chromatography (TLC) was performed using Merck Silica gel 60 F₂₅₄ and RP-18 F₂₅₄ plates. All reagents were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).