The compounds separated by LC were analyzed in both positive- and negative-ion modes. The mass spectrometer was operated in the positive and the negative ESI modes with a Duo Spray TM source (AB SCIEX, Canada) connected to an MS/MS (Triple TOF 5600, AB SCIEX). The following parameter settings were used: ion spray voltage, ±5,500 V; ion source temperature, 600°C; curtain gas, 30 psi; both ion source gas 1 and gas 2, 55 psi; TOF-MS scan, m/z 100–1,000 Da of mass range; and scan accumulation time, 0.25 s/spectra. In high sensitivity mode, they were as follows: ion scan m/z range, 50–1,000 Da; ion scan accumulation time, 0.07 s/spectra; declustering potential, ±60 V; and collision energy, ±20 V. In addition, information-dependent acquisition (IDA) was used to acquire MS/MS spectra for ions matching the IDA criteria (excluding isotopes within 4 Da, candidate ions to monitor per cycle: 10).
Duo spray tm
Manufactured by AB Sciex
Sourced in Canada
The Duo Spray TM is a specialized laboratory equipment designed for liquid sample introduction in mass spectrometry analysis. It features dual independent nebulizers that allow simultaneous introduction of two separate liquid samples into the mass spectrometer. The core function of the Duo Spray TM is to enable efficient and accurate sample delivery for various analytical applications.
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3 protocols using duo spray tm
1
Targeted Metabolite Analysis by LC-MS/MS
2
Phytoconstituent Separation and Identification
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Separation of phytoconstituents was performed on an Axion AC system (Kyoto, Japan) connected with an auto sampler system, an in-line filter disks pre column (0.5 µm × 3.0 mm, Phenomenex, Torrance, CA, USA), and a Xbridge C18 (3.5 µm, 2.1 × 50 mm) column (Waters Corporation, Milford, MA, USA) [25 (link)]. Mass spectrometry was performed on a Triple TOFTM 5600 system quadrupole-time-of-flight mass spectrometer (QTOF/MS) with a Duo-Spray TM source operating in the ESI mode (AB SCIEX, Concord, Toronto, ON, Canada), with sprayer capillary and de-clustering potential voltages of 4500 and 80 V, respectively. The method consisted of high-resolution survey spectra at which mobile phase composition, gradient elution, source temperature, collision energy, ion tolerance, operation parameters, and the database search parameters’ settings were performed according to the method discussed in our previous publication [25 (link)].
3
Nano-HPLC-MS/MS Peptide Separation
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Peptides were chromatographically separated using an Ultimate 3000 nano-high performance liquid chromatography (HPLC) system (LC Packings, DIONEX, Thermo Fisher Scientific, Sunnyvale, CA, USA). Samples were pre-concentrated in a pre-column cartridge (PepMap-100 C18 5 mm 100 A, 30 mm id  5 mm, LC Packings) by the loading pump. Chromatographic separation of peptides was performed using a C18 PepMap-100 column (15 cm  75 mm id, LC Packings) equilibrated at 30 8C with a solvent A (water/acetonitrile 98/2 (vol/vol), 0.1% formic acid) at a flow rate of 300 nL min À1 . Runs were performed under 60 min linear gradient from 10% to 45% of solvent B (water/acetonitrile 2/ 98 (vol/vol), 0.1% formic acid) followed by 10 min of a purge step and 20 min re-equilibration step. The HPLC system was directly coupled to TripleTOF TM 5600 System (AB SCIEX, Toronto, Canada), equipped with a DuoSpray TM ion source (AB SCIEX). Eluted peptides were directly processed through the mass spectrometer, controlled by Analyst 1 1.6.1 software (AB SCIEX). For information dependent acquisition (IDA) analysis, survey scans were acquired in 250 ms and 25 product ion scans exceeding a threshold of 125 counts per second (counts/s) were collected. Dynamic exclusion was set for 1/2 of peak width ($8 s), and then the precursor was refreshed off the exclusion list.
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