6520 accurate mass q tof lc ms

Peptide characterization for the proteolyzed almond extract was performed on an Agilent 6520 Accurate-Mass Q-TOF LC-MS. The peptide samples purified by reverse-phase or mixed-mode SPE were injected into an Agilent Zorbax 300SB-C18 chip (40 nL enrichment column, 75 μm × 150 mm; for comparing different protein removal approaches) or an Agilent Polaris-HR-Chip (360 nL enrichment column, 75 μm × 150 mm; for comparing different SPE approaches) and separated by a mobile phase, consisting of 3% ACN with 0.1% formic acid (A) and 89.9% ACN with 0.1% formic acid (B), eluted at a flow rate of 300 nL min⁻¹ with the following gradients: 0–2.3% B from 0–0.1 min; 2.3–8% B from 0.1–2.0 min; 8–37% B from 2.0–40.0 min; 37–48 % B from 40.0–45.0 min; 48–100% B from 45.0–45.1 min; 100% B from 45.1–50.0 min; 100–0% B from 50.0–50.1 min; and 0% B from 50.1–65.0 min. The scan range was m/z 70–1800 for MS and m/z 50–1800 for MS/MS. The scan speed was set at 8 spectra sec⁻¹ for MS and varied with precursor abundance with a target of 25,000 count spectrum⁻¹ for MS/MS, respectively. The ESI source was operated on positive mode with a capillary voltage of 1950 V and drying gas at 325 °C and 5 L min⁻¹. The top 10 ions with the highest intensities in each cycle were selected for tandem MS analysis with the CE set by a formula of (CE (V) = 0.03 × (m/z) + 2).

Nuclear magnetic resonance (NMR) spectra were recorded on either a Varian INOVA 600 (600/150 MHz) or Varian INOVA 500 (500/125 MHz) spectrometer. Chemical shifts are quoted in parts per million (ppm) relative to tetramethylsilane standard and with the indicated solvent as an internal reference. The following abbreviations are used to describe signal multiplicities: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), dd (doublet of doublets), dt (doublet of triplets), etc. Accurate mass spectra were recorded on an Agilent 6520 Accurate-Mass Q-TOF LC/MS.
Non-aqueous reactions were performed under an atmosphere of argon, in flame-dried glassware, with HPLC-grade solvents dried by passage through activated alumina. Brine refers to a saturated aqueous solution of sodium chloride, sat. NaHCO₃ refers to a saturated aqueous solution of sodium bicarbonate, sat. NH₄Cl refers to a saturated aqueous solution of ammonium chloride, etc. “Column chromatography,” unless otherwise indicated, refers to purification in a gradient of increasing ethyl acetate concentration in hexanes or methanol concentration in dichloromethane on a Biotage^® flash chromatography purification system. All chemicals were used as received from Oakwood, TCI America, Sigma-Aldrich, Alfa Aesar, or CombiBlocks.

The band of interest was excised from the gel and digested using 15 μL of 5 ng/μL trypsin (V5111, Promega) in 25 mM ammonium bicarbonate overnight at 37°C. The peptide digest was extracted from the gel with 5% acetonitrile (ACN), 2% formic acid (FA) in water. Then 8 μL of the extract was subjected to LC-MS/MS analysis on a 1200 HPLC-Chip system coupled with the 6520 Accurate-Mass Q-TOF LC-MS (Agilent Technologies, California, US). A HPLC-Chip with ZORBAX 300SB-C18 (300Å), 5 μm particles, 40 nL enrichment column, and a 75 μm x 43 mm analytical column was used. Mobile phases were mixtures of ACN, water and FA. A 20 minute linear gradient from 5–40% ACN was used for the separation on the analytical column. The MS/MS spectra were converted to peak lists in the Mascot generic format (mgf) and used in a SwissProt database search with MS/MS Ion Search (Mascot, Matrix Sciences). Database search parameters included: enzyme: trypsin, variable modification: oxidation of methionine, peptide mass tolerance: ± 10 ppm and fragment mass tolerance: ± 0.3 Da.

Approximately 0.2 g of ground seed coat or kernel and 1.5 mL of extract solution (methanol:water = 70:30, v/v) were added into a 5 mL centrifuge tube and placed in an ultrasonic cleaner at 20 °C for 20 min. The supernatant was then transferred into another tube after centrifugation. The above procedure was repeated twice. The extract was filtered through a membrane (0.22 μm) and stored at −40 °C for further analysis.
For further HPLC analysis, the following solvent and gradient were used: A, 2% aqueous formic acid (v/v); B, 0.2% formic acid in acetonitrile (v/v); constant gradient from 5% to 64% of B within 80 min; a flow rate of 0.6 mL/min; and 10 μL of extract solution, injected for detection. The column temperature was maintained at 30 °C. DAD data were recorded at 280 nm.
An Agilent 6520 Accurate-Mass Q-TOF LC/MS was used for the qualitative analysis of the compounds. The electrospray ionization mass spectrometry method was applied with positive or negative ion modes. The scanning range (m/z) was 100–1200 u, the sprayer pressure was 35 psi, and the capillary voltage was 3500 V. Dry gas at 350 °C was carried at a flow rate of 12 L/min. The data were analyzed by Masshunter Qualitative Analysis Software B. 04. 00.