Tripletof 6600 mass spectrometry

The untargeted metabolomic analysis was performed by utilizing a 1,290 Infinity series UHPLC System (Waters Corporation, Milford, MA, USA). The mobile phase was composed of 25 mmol/L ammonium acetate in water was applied as phase A, and 25 mmol/L ammonia in acetonitrile was used as phase B. The analysis procedure was processed as previously described (Zhao et al., 2019 (link)). The Triple TOF 6600 mass spectrometry (AB Sciex, Boston, MA, USA) was used to obtain spectra data, and the acquisition software (Analyst TF 1.7, AB Sciex, Framingham, MA, USA) continuously evaluated the full-scan survey MS data. In each cycle, the most intensive 12 precursor ions (intensity > 100) were chosen for MS/MS at collision energy (CE) of 30 eV. The cycle time was 0.56 s. Electrospray ionization (ESI) source conditions were set as previous study described (Liu et al., 2016 (link); Zhao et al., 2019 (link)).

The UHPLC separation was carried out using a 1,290 Infinity series UHPLC System (Agilent Technologies), equipped with a UPLC BEH Amide column (2.1 * 100 mm, 1.7 μm, Waters). The analysis was performed using elution gradient as follows: 0 ∼ 0.5 min, 95% B; 0.5 ∼ 7.0 min, 95 ∼ 65% B; 7.0 ∼ 8.0 min, 65%–40% B; 8.0–9.0 min, 40% B; 9.0 ∼ 9.1 min, 40 ∼ 95% B; 9.1 ∼ 12.0 min, 95% B. The mobile phase consisted of 25 mmol/L ammonium acetate and 25 ammonia hydroxide in water (pH = 9.75) 1) and acetonitrile 2) and the column temperature was 25°C. The autosampler temperature was 4°C, and the injection volume was 1 μL (POS) and 1 μL (NEG).
During the LC–MS experiment, the TripleTOF 6,600 mass spectrometry (AB Sciex) was utilized to obtain MS–MS. The software (Analyst TF 1.7, AB Sciex) endlessly estimates the complete scan survey MS data for collecting and activating the acquirement of MS/MS spectra subject upon preselected criteria. For the individual cycles, an MS/MS at collision energy (CE) of 30 eV was used with preselected 12 ions above 100 intensity. Every cycle lasted 0.56 s. The source conditions for the ESI were established according to the following: gas 1 at 60 psi, gas 2 at 60 psi, curtain gas at 35 psi, the temperature at 600°C, declustering potential at 60 V, ion spray voltage floating at 5000 V in positive and −4000 V in the negative mode.

Samples were analyzed on a 1290 Infinity series UHPLC System (Waters Corporation, Milford, MA, USA) as previous study described [17 (link)]. Briefly, 10 μL of reconstituted sample was injected on a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm). The mobile phase consisted of 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide in water (pH = 9.75) (A) and acetonitrile (B). Each sample was analyzed in the positive ion mode and negative ion mode. The Triple TOF 6600 mass spectrometry (AB Sciex, Boston, MA, USA) was used for its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during an LC/MS experiment. In this mode, the acquisition software (Analyst TF 1.7, AB Sciex, Framingham, MA, USA) continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on the preselected criteria. In each cycle, the most intensive 12 precursor ions with intensity above 100 were chosen for MS/MS at a collision energy (CE) of 30 eV. The cycle time was 0.56 second. Electrospray ionization (ESI) source conditions were set as follows: gas 1 as 60 psi, gas 2 as 60 psi, curtain gas as 35 psi, source temperature as 600°C, declustering potential as 60 V, and ion spray voltage floating (ISVF) as 5000 V or -4000 V in the positive or negative modes, respectively.

Plasma samples of patients were prepared for ultra-high-performance liquid tandem chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS) analysis by application of validated protocols (Dunn et al., 2011 (link)). The UHPLC separation was carried out using a 1290 Infinity series UHPLC System (Agilent Technologies Inc., Santa Clara, California, United States), equipped with a UPLC BEH Amide column. The TripleTOF 6600 mass spectrometry (AB Sciex, Foster City, CA, United States) was used for its ability to acquire tandem mass spectrometry spectra on an information-dependent basis during a liquid chromatography–mass spectrometry experiment. Both positive ion mode (POS) and negative ion mode (NEG) were used to obtain maximal coverage for plasma metabolites.

UHPLC separation was carried out using a 1290 Infinity series UHPLC System (Agilent Technologies) equipped with a UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm, Waters). The mobile phase consisted of 25 mmol/l ammonium acetate and 25 ammonia hydroxide in water (pH = 9.75) (A) and acetonitrile (B). The analysis was carried out with an elution gradient as follows: 0–0.5 min, 95% B; 0.5–7.0 min, 95–65% B; 7.0–8.0 min, 65–40% B; 8.0–9.0 min, 40% B; 9.0–9.1 min, 40–95% B; 9.1–12.0 min, 95% B. The column temperature was 25 °C. The autosampler temperature was 4 °C, and the injection volume was 1 μl (pos) or 1 μl (neg).
TripleTOF 6600 mass spectrometry (AB Sciex) was used to acquire MS/MS spectra on an information-dependent basis (IDA) during an LC–MS experiment. In this mode, acquisition software (Analyst TF 1.7, AB Sciex) continuously evaluated the full scan survey MS data as it collected and triggered the acquisition of MS/MS spectra depending on preselected criteria. In each cycle, the 12 most intensive precursor ions with intensities. 100 were chosen for MS/MS at a collision energy (CE) of 30 eV. The cycle time was 0.56 s. ESI source conditions were set as follows: gas 1, 60 psi; gas 2, 60 psi; curtain gas, 35 psi; source temperature, 600 °C; declustering potential, 60 V; ion spray voltage floating (ISVF), 5000 V and − 4000 V in positive or negative modes, respectively [19 (link)].

Hesperidin and vitex were analyzed as described previously with some modifications (Miura et al., 2020). A 200 mg sample of powder was weighed and extracted overnight at 4°C in 1.0 ml of 70% aqueous methanol. Following centrifugation at 10,000 × g for 10 min, the extracts were absorbed (CNWBOND Carbon‐GCB SPE Cartridge, 250 mg, 3 ml; ANPEL) and filtered (SCAA‐104, 0.22 µm pore size; ANPEL) before LC‐MS analysis. The HPLC conditions were as follows: HPLC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 µm, 2.1 mm × 100 mm), solvent system, water: acetonitrile, gradient program, 90:10 v/v at 0 min, 90:10 v/v at 1.0 min, 10:90 v/v at 3 min, 10:90 v/v at 5 min, 10:90 v/v at 6 min; flow rate, 0.42 ml/min; temperature, 40°C; and injection volume: 2 µl. The TripleTOF 6,600 mass spectrometry (AB Sciex) was used for its ability to acquire MS/MS spectra on an information‐dependent basis (IDA) during an LC/MS experiment. The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 500°C; ion spray voltage (IS) 5,500 V; ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively.

For each sample, 1 × 10⁷ cells were collected from a 20 cm plate and quickly frozen in liquid nitrogen. Six independent samples were prepared for each group. 1000 μL extract solution (acetonitrile: methanol: water = 2: 2: 1) containing an internal standard was added, and the samples were homogenized and sonicated. After incubation at -40 ℃ for 1 h, the sample were centrifuged with 12,000 rpm for 15 min. Then, 800 μL of the supernatant was transferred to a fresh tube and dried in a vacuum concentrator. The dried samples were reconstituted in 100 μL acetonitrile solution and subjected to 10 min sonication. After centrifugation at 13,000 rpm for 15 min, 75 ul of the supernatant was transferred to a fresh glass vial for LC–MS analysis. The LC–MS/MS analysis of HPNE cells, PANC-1 cells and Capan-1 cells was conducted using a 1290 Infinity series UHPLC System (Agilent Technologies) and TripleTOF 6600 mass spectrometry (AB Sciex) by BIOTREE BIOMEDICAL TECHNOLOGY CO., LTD (Shanghai, China). The global metabolite profiling of PRM cells and LMT cells was accomplished using an LC–ESI–MS/MS system (UPLC, ExionLC AD; MS, QTRAP® System) was accomplished by Metware Biotechnology Inc (Wuhan, China).