6520 quadrupole time of flight mass spectrometer

Proteomic analysis of digested Naja haje fraction was carried out in an Agilent 1200 Series HPLC-Chip/MS system connected to a 6520 Quadrupole-Time of Flight (Q-TOF) mass spectrometer, equipped with a nano-electrospray source (Agilent Technologies, Santa Clara, CA, USA). For the online fractionation, two microliters of tryptic peptides were loaded and enriched on a 160 nL RP-C18 trap column, then separated on an analytical nano-column (150 mm × 75 µm) packed with ZORBAX SB-C18, 5 µm, 300Å (G4240-62010; Agilent Technologies). The separation was maintained over 25 min at 450 nL/min, using a linear gradient from 3 to 80% ACN in 0.1% FA. The eluted peptides were operated in positive mode in the Q-TOF mass spectrometer; the mass range was set from 290 to 1700 m/z and from 59 to 1700 for the MS and MS/MS scans, respectively. The total cycle time was two seconds. In each cycle, five of the most abundant precursor ions were subjected to MS/MS fragmentation; the ions with single charge were excluded, while the collision energy was automatically adjusted according to m/z [44 (link)].

The full spectrum of modified ribonucleosides in the purified 5S rRNA was characterized by analysis of ribonucleoside hydrolysates of the RNA by chromatography-coupled quadrupole time-of-flight and triple quadrupole mass spectrometry, using methods described previously (15 (link),26 (link),27 (link)). Briefly, purified BCG 5S rRNA was enzymatically hydrolyzed in the presence of antioxidants and deaminase inhibitors and the ribonucleosides resolved by HPLC using a Hypersil GOLD aQ Analytical HPLC Column (100 × 2.1 mm, 1.9-μm particle size) (Thermo Scientific, Wilmington, DE, USA) coupled to either an Agilent 6460 triple quadrupole mass spectrometer or an Agilent 6520 quadrupole time-of-flight mass spectrometer (LC-MS/MS) with ESI ionization operated in positive ion mode. The identity of individual ribonucleosides was confirmed by comparison with synthetic standards for HPLC retention time, exact molecular weight and collision-induced dissociation (CID) fragmentation patterns. In cases where a standard was not available, a putative structure was inferred from exact molecular weight and CID fragmentation patterns.

The fermentation process and metabolite extraction of M. purpurea was done based on previously described protocols [21 ]. Its antibiotic activity was detected by the cylinder-plate method, which was done according to Pharmacopoeia of the People’s Republic of China (2010 edition) [22 ]. The components of the product were analyzed by thin layer chromatography (TLC) using silica gel GF254 plate. The development solvent used in the assay was the underlayer of the mixture (chloroform: methanol: ammonia in 1:1:1 volume ratio). Accurate component ascertainment was performed by electrospray ionization mass spectrometry (ESI/MS) using Agilent 6520 quadrupole-time-of-flight mass spectrometer. The scanning range of Q-TOP-MS is set as following: Positive ion mode m/z 100~800; the dry gas is N₂; the flow rate is 8mL/min; the temperature is 350 °C; atomization pressure is 2.07 × 10⁵ Pa; capillary voltage is 3500 V; the fragmentation voltage is 135 V; and Agilent MassHunter is used to analysis MS data (B.04.00).

Online HPLC-QTOF-MS analysis was made using an Agilent 1290 infinity LC system connected to a 6520 quadrupole time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray interface. The positive and negative ion ESI-MS experiments were conducted using conditions as follows: drying gas temperature, 320°C; drying gas (N₂) flow rate, 10 l/min; nebulizer, 45 psi; capillary voltage, 4000 V for positive mode and 3500 V for negative mode; and fragmentor, 175 V. All the operation, acquisition, and analysis of data were made by Agilent Mass Hunter Acquisition Software Version B.04.00 (Agilent Technologies).

Liquid chromatography was performed on an Agilent Series G2226A pump by injecting 5 μL sample onto an Agilent Zorbax SB-Aq Rapid Resolution HT 2.1 x 100 mm, 1.8 μm, 600 bar analytical column, coupled to an Agilent Zorbax SB-Aq Narrow-Bore 2.1 x 12.5 mm, 5 μm guard column. The autosampler tray temperature was set at 4 °C and column compartment was set at 30 °C. Samples were run at a flow rate of 0.3 mL/min, using mobile phase A (water with 0.1% formic acid), and mobile phase B (90:10 acetonitrile:water with 0.1% formic acid). Gradient elution was as follows: 0-3 min 2% B, 3-5 min 2-40% B, 5-20 min 40-100% B, 20-30 min 100% B, followed by column re-equilibration. An Agilent 6520 quadrupole time-of-flight mass spectrometer (Q-TOF-MS) was used to analyze samples in positive and negative ionization mode at mass range 50-1700 m/z, scan rate 2.22 spectra/s, gas temperature 300^oC, and gas flow 10 L/min. The nebulizer was 30 psi, skimmer 60 V, capillary voltage 4000 V, and fragmentor 120 V in positive mode and 140 V in negative mode, with reference masses 121.050873 and 922.009798 for positive mode and 112.985628 and 966.000725 for negative mode (Agilent reference mix).

Samples (both in vivo and in vitro) were centrifuged at 14,000× g rpm for 10 min at 4 °C, and the supernatants were transferred to glass insert liquid chromatography vials. Analyses occurred on an Agilent 1290 liquid chromatography system coupled to an Agilent 6520 quadrupole time of flight mass spectrometer. Samples (5 µL) were injected and separated on a Waters Acquity UPLC BEH (bridged ethyl hybrid) Amide 1.7 µm 2.1 × 100 mm HILIC (hydrophilic interaction liquid chromatography) column with a flow rate of 0.3 mL/minute. Mobile phases consisted of A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid). The column was equilibrated at 2.5/97.5 (A/B) and maintained for 1 min post injection. Mobile-phase A increased in a linear gradient from 2.5% to 65% from 1 to 9 min post injection then stepped to 97.5% A from 9 to 11 min to wash the column. Column was equilibrated in starting condition for 3 min before the next injection. The mass spectrometer, equipped with a dual electrospray ionization source, was run in negative ion and then in positive ion mode. The scan range was 50–1600 m/z. The source settings consisted of drying gas flow rate: 11 L/min; nebulizer: 40 pounds per square inch gauge; gas temp: 350 °C; capillary voltage: 3000 V (neg), 2500 V (pos).