For CZE-MS/MS, the proteolysis products of free mSA, native GlpG, and denatured GlpG were obtained using the solid phase–enhanced method (86 (link)). After washing with 90% acetonitrile and elution with 100 mM NH4HCO3 buffer (pH 8), recovered peptides were separated by CZE (the ECE-001 system, CMP Scientific). For RPLC-MS/MS, RPLC was performed on a Vanquish Flex UPLC system (Thermo Fisher) and an Acquity BEH-C4 column (Waters) using a 1 to 99% acetonitrile gradient in water containing 0.1% formic acid. For mass detection, a Q-Exactive HF MS/MS spectrometer (Thermo Fisher) was used with a scan range of 200 to 2,000 m/z. The data were analyzed on Byonic (version 3.9.6, Protein Metrics) (87 ).
Vanquish flex uplc system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Vanquish Flex UPLC system is a high-performance liquid chromatography (HPLC) instrument designed for ultra-high-performance liquid chromatography (UHPLC) applications. It provides fast and efficient separation of complex samples, enabling improved resolution and sensitivity for a wide range of analytical applications.
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Lab products found in correlation
Acquity beh c4 column, by Waters Corporation (1 mentions)
Byonic, by Protein Metrics (1 mentions)
Acquity uplc t3 column, by Waters Corporation (1 mentions)
Ltq velos pro linear ion trap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Beh c18 column, by Waters Corporation (1 mentions)
Acquity hss t3 column, by Thermo Fisher Scientific (1 mentions)
4 protocols using vanquish flex uplc system
1
Comparative Proteolysis Analysis by CZE-MS/MS and RPLC-MS/MS
2
Phytochemical Profiling of Fruit Extracts
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The phytochemical profile of the crude extracts of the peel, flesh, and seeds was assessed by LC-MS/MS analysis utilizing the reported method Asghar et al. [19 (link)] The chromatographic separations were carried out using a Vanquish Flex UPLC system (Thermo, Germany), while an ACQUITY UPLC T3 column (100 mm × 2.1 mm, 1.8 μm, Waters-Milford, USA) was used for the reversed-phase separation. The Q-Exactive was utilized in positive and negative ion modes, and MS-DIAL software was employed to extract the peaks and identify metabolites from the raw data. Additionally, the secondary mass spectrogram data from the sample experiment was used to compare to the METLIN database, and the molecules were selected that matched the Molecular Formula Generator (MFG) score of ≥86 %.
3
UPLC-MS/MS Quantification of Bioactive Compounds
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Vasicine, vasicinone, isoorientin, isovitexin, and apigenin were quantified using a Thermo Vanquish Flex UPLC System interfaced with an LTQ Velos Pro Linear Ion Trap Mass Spectrometer (ThermoFisher, San Jose, CA, USA). Chromatographic separation of the analytes was achieved using a BEH C18 column (150 × 3 mm i.d.; 1.7 μm particle size; Waters, Milford, MA) at 50 °C with gradient elution. The mobile phases were (A) 0.1% formic acid in H2O and (B) methanol and a flow rate of 300 mL/minute. The percentage of B varied as follows: 2% B at 0 min, 2%→95% (0 to 7.5 min), hold at 95% (7.5 to 8.5 min), 95%→2% (8.5 to 8.6 min), and hold at 2% from 8.6 to 10 min. A convex gradient (setting = 6 during the 0–7.5 min ramp) was used. The mass spectrometer was operated in positive electrospray ionization, selective reaction-monitoring mode. The MS/MS parameters were optimized by infusion of vasicine into the MS/MS system and the following precursor→product transitions were used: m/z 189.1→m/z 171.1 for vasicine, m/z 203.2→m/z 185.1 for vasicinone, m/z 449.1→m/z 431.1 for isoorientin, m/z 433.2→m/z 415.1 for isovitexin, and m/z 271.1→m/z 271.1 + 243.1 for apigenin. Data acquisition and processing were performed using Xcalibur software, and calibration curves were prepared in water using analyte peak area versus concentration.
4
UPLC-MS/MS Analysis of Compounds
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UPLC separation was performed using a Vanquish Flex UPLC system (Thermo Fisher Scientific, USA) on a ACQUITY HSS T3 column (2.1 × 150 mm, 1.8 μm) at a column temperature of 35°C. The mobile phase was water (0.1% formic acid, A) and acetonitrile (0.1% formic acid, B). The UPLC system was eluted with a gradient program as follows: 10–90% B at 0–25 min, 90 − 10% B at 25–25.1 min, 10% B at 25.1–30 min. The flow rate was 0.3 mL/min.
MS analysis was performed using an Orbitrap Fusion Lumos tribrid mass spectrometer equipped with a heating electrospray ionization source (ESI). The following ESI source parameters were used: an ion spray voltage of 3.2 kV, a capillary temperature of 350°C, an ion transfer tube temperature of 320°C, a sheath gas (N2) flow rate of 42 arb, a sweep gas (N2) flow rate of 1 arb, and an auxiliary gas (N2) flow rate of 12 arb. MS spectra were acquired at the mass range of 350–2000 m/z. High collision-induced dissociation (HCD) was adopted with normalized collision energy setting of 40 eV in ESI− mode and 20 eV in the ESI+ mode. MS2 spectra were acquired by the data-dependent acquisition (DDA) scan mode, and the primary ions with ionic strength greater than 2.5e4 were broken into secondary fragments. Dynamic exclusion was set to 6.00 s.
MS analysis was performed using an Orbitrap Fusion Lumos tribrid mass spectrometer equipped with a heating electrospray ionization source (ESI). The following ESI source parameters were used: an ion spray voltage of 3.2 kV, a capillary temperature of 350°C, an ion transfer tube temperature of 320°C, a sheath gas (N2) flow rate of 42 arb, a sweep gas (N2) flow rate of 1 arb, and an auxiliary gas (N2) flow rate of 12 arb. MS spectra were acquired at the mass range of 350–2000 m/z. High collision-induced dissociation (HCD) was adopted with normalized collision energy setting of 40 eV in ESI− mode and 20 eV in the ESI+ mode. MS2 spectra were acquired by the data-dependent acquisition (DDA) scan mode, and the primary ions with ionic strength greater than 2.5e4 were broken into secondary fragments. Dynamic exclusion was set to 6.00 s.
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