X500r q tof mass spectrometer

Prior to the HPLC-ESI-MS analysis, the lyophilized mixture was rehydrated with 1.0 mL of Milli-Q water. Before being used, the water was boiled for 5 min and then cooled to 4 °C. The rehydrated solution was stored at −20 °C until analysis.
HPLC-ESI-MS was carried out on a SCIEX X500R Q-TOF mass spectrometer (Framingham, U.S.A.). The MS conditions were as follows: ESI-MS analysis was performed using a SCIEX X500R Q-TOF mass spectrometer equipped with an ESI source. The mass range was set at m/z 100–1000. The Q-TOF MS data were acquired in the positive mode, and the conditions of MS analysis were as follows: CAD gas flow-rate, 7 L min⁻¹; drying gas temperature, 550 °C; ion spray voltage, 5500 V; declustering potential, 80 V. Software generated data file: SCIEX OS 1.0.

The LJP-A-3-5-3 fraction was collected. Subsequently, LJP-A-3-5-3 was separated by RP-HPLC (Agilent 1200) on a Zorbax SB C-18 column (4.6 × 250 mm, 5 µm). The elution solvent system was composed of water-trifluoroacetic acid (solvent A; 100:0.1, v/v) and acetonitrile-trifluoroacetic acid (solvent B; 100:0.1, v/v). The peptides were separated using a 30% to 70% gradient elution of solvent B for 45 min at a flow rate of 1.0 mL/min, with the detection wavelength set at 280 nm.
HPLC-ESI-MS was performed on a SCIEX X500R Q-TOF mass spectrometer (Framingham, MA, USA). The MS conditions were as follows: ESI-MS analysis was performed using a SCIEX X500R Q-TOF mass spectrometer equipped with an ESI source. The mass range was set to m/z 100–1200. The Q-TOF MS data were acquired in positive mode, and the MS analysis conditions were as follows: CAD gas flow rate, 7 L/min; drying gas temperature, 550 °C; ion spray voltage, 5500 V; declustering potential, 80 V; software-generated data file: SCIEX OS 1.0. The peptide is usually protonated under ESI-MS/MS conditions, and fragmentations occur mostly at the amide bonds because it is difficult to break the chemical bonds of the side chains at such low energy. Hence, on the basis of the HPLC-ESI-MS data and accompanied by the Edman degradation results, the amino acid sequences of the LJPs were identified.

LC-MS was performed using an AB SCIEX X500R Q-TOF mass spectrometer with the AB SCIEX X500R SCIEX OS software (AB SCIEX, USA). Mass spectrometry (MS) was conducted using an electrospray ionization (ESI) ion source in the negative ion mode. The scanning range was set to 100–1200 Da, the capillary voltage was −4500 V, and the ion source temperature was 600 °C. The other optimized conditions of the ESI source were as follows: cone hole gas flow rate, 50 L/h; nebulizing gas, 500 L/h; drying gas (Gas1), 55 psi; gas curtain gas (Gas2), 55 psi; collision gas, 7 psi; declustering voltage, 80 V; scan time, 0.52 s; cumulative sampling time, 0.1 s.
Chromatography was performed using an ACQUITY UPLC (Waters, USA) system with an ACQUITY UPLC T3 C18 column (100 × 2.1 mm, 1.8 μm) at 30 °C. The mobile phase consisted of 0.1% formic acid water solution (A) and methanol (B) in the gradient elution mode as follows: 6–17% B, 0–2 min; 17–26% B, 2–15 min; 26–32% B, 15–25 min; 32–38% B, 25–40 min; 38–48% B, 40–50 min; 48–6% B, 50–55 min. The flow rate was 0.3 mL/min, the sample injection volume was 1 μL, and the detection wavelength was set to 270 nm.

NMR, UV, and IR spectra were obtained using Bruker AV-600 (Bruker; Ettlingen, Germany), JASCO V-550 UV-VIS (Jasco; Tokyo, Japan), and JASCO FT/IR-480 plus FT-IR spectrometers (Jasco; Tokyo, Japan), respectively. LC-QTOF-MS data were generated using a Shimadzu LC-20AD liquid chromatogram (Shimadzu; Kyoto, Japan) and an AB SCIEX X500R QTOF mass spectrometer (AB SCIEX; Framingham, MA, USA). The MTT reagent was obtained from Keygen Biotech located in Nanjing, China. The PI reagent was obtained from Sigma-Aldrich^®, (St. Louis, MO, USA). The RNase A reagent was procured from Fermentas^® (Shanghai, China). Detailed instruments and chemicals are provided in the Supporting Information.

Mass spectrometry was performed on an X-500R QTOF mass spectrometer (AB Sciex LLC, Framingham, USA). Instrument handling and data acquisition were performed using SCIEX OS Software (version 2.2, AB Sciex LLC, Framingham, USA). Data processing, such as extraction of extracted-ion chromatograms, calculation of mean spectra, and baseline chromatogram extraction, was performed using the built-in Explorer Tool. A peak width of m/z ± 0.02 was used for all molecules to extract extracted-ion chromatograms. The baseline chromatogram was extracted with an m/z ratio between 400 and 500. Protein reconstructions from spectra were performed using the Bio Tool Kit of the Explorer Tool with a limited input m/z range from 1700 to 2600. The output mass range was chosen from 5 to 20 kDa with a step mass of 0.5 Da.