An LC instruments set (Agilent Technologies 1260 Infinity II, Santa Clara, CA, USA) was connected to a time-of-flight mass spectrometer (TOFMS; Agilent Technologies G6230B) with an electrospray ionization source (Dual Agilent Jet Stream ESI). TOFMS detection was operated in negative ionization mode using the following scan source parameters: capillary voltage 3.5 kV, ion source gas temperature 300 °C, drying gas flow 10 L/min, and sheath gas temperature 350 °C with sheath gas flow 11 L/min. The column used was ZORBAX Extend-C18 (2.1 × 50 mm, particle size 1.8 μm; Agilent Technologies, P/N: 727700-902), and the column temperature was set at 45 °C. Column equilibration was carried out 1 day before actual measurement by passing mobile phases A and B at the starting ratio according to each measurement method.
Jet stream esi
Manufactured by Agilent Technologies
Sourced in United States
The Jet Stream ESI is an electrospray ionization (ESI) source for mass spectrometry applications. It provides a stable and efficient ionization process to enable the analysis of a wide range of analytes. The core function of the Jet Stream ESI is to generate charged droplets from the sample solution, which are then transported into the mass spectrometer for further analysis.
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4 protocols using jet stream esi
1
Quantitative LC-TOFMS Analysis Protocol
2
Identification of Unknown Selenium Metabolites
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An Agilent 6320 ESI-ion trap-MS was operated in the negative-ion mode. The fraction collected from IC containing the unknowns was directly introduced into the electrospray source using a syringe pump (KDS 100CE; KD Scientific, Holliston, MA, USA) (see the supplemental material). Unknown Se metabolites were recognized by the Se isotopic pattern in full-scan mass spectra. High-resolution MS was done on an Agilent 6540 UHD mass spectrometer equipped with Agilent Jet Stream ESI and operated in negative mode (see Table S3). The collected peak fraction of unknown1 was introduced into the ESI using the syringe pump at a speed of 5 μl/min. Data were processed by Agilent MassHunter Data Acquisition for 6500 series Q-TOF-MS (B.08.00). The possible chemical structures of unknown1 were generated based on accurate mass, isotope abundance, and isotope patterns using the “formula calculator” tool in Agilent MassHunter Qualitative Analysis (B.06.00 SP 1) software.
3
Characterizing mAb Deglycosylation via RPLC-MS
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mAb deglycosylation was performed as discussed in Sect. 2.2 , followed by RPLC using the Advance Bio RP C8 column (Agilent Technologies) operated at 80 °C on the Agilent Infinity LC system consisting of a quaternary pump with a degasser, an auto sampler with a cooling unit and a diode array detector (DAD) coupled to an ESI–QTOF–MS (Agilent 6550 iFunnel Q-TOF–LC–ESI–MS). Before injection, the column was saturated with 90% mobile phase A (0.1% v/v formic acid (FA) in MilliQ) and 10% mobile phase B (0.1% v/v FA in acetonitrile). The sample (2 µl of 1 mg/ml) was loaded onto the column and separated with a 10–70% gradient of 10–70% of solvent B at a flow rate of 0.5 ml/min for 30 min. Detection was carried out by monitoring UV absorption at 280 nm and TIC was recorded for 1000–7000 m/z. The MS parameters applied included capillary voltage 4000 V; sheath gas flow 12, sheath gas temperature 280 °C, gas flow (l/m) 13. A total of 220 MS spectra were calibrated in the positive ion mode and deconvoluted using maximum entropy (MaxEnt) as part of Agilent Mass Hunter Qualitative Analysis (Nupur et al. 2018 (link)). MS calibration of the Agilent 6550 iFunnel instrument coupled with a dual Agilent Jet Stream ESI was performed using tuning mix (#G1969-85000 ESI-L).
4
UPLC-MS/MS for Targeted Metabolite Analysis
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A chromatographic separation was performed on a Waters ACQUITY UPLC BEH HILIC (130 Å, 2.1× 100 mm, 1.7 μm) column, thermostatted at 60 °C in an Agilent 1290 UHPLC system. The flow rate was 400 μl min−1 with mobile phase A composed of acetonitrile and 25 mM ammonium formate buffer (50:50, v/v) and mobile phase B composed of acetonitrile and 25 mM ammonium formate buffer (95:5, v/v). Mobile phases A and B were mixed to create the following gradient: 99.90% B at 0 min to 40% B at 5.00 min and 10% B at 6.50 min and reequilibrated at 99.90% B from 6.60 min to 9.60 min. The total run time was 9.60 min. An Agilent 6495 QQQ was used for targeted measurements. The Agilent Jet Stream ESI source parameters for the MS were dry gas temperature and flow of 200 °C and 12 l min−1, respectively, nebulizer pressure 25 psi, sheath gas temperature and flow were set to 400 °C and 12 l min−1, respectively, capillary voltage and nozzle voltage set to 3500 V and 500 V, respectively, and the delta electron multiplier voltage of 200 V. Positive high/low pressure radio frequency was set to 200/110.
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