6530 esi qtof mass spectrometer

Reversed-phase chromatography
was performed in-line prior to mass spectrometry using an Agilent
1290 uHPLC system (Agilent Technologies inc. USA). Concentrated protein
samples were diluted to 0.02 mg/mL in 0.1% formic acid and 50 μL
was injected on to a 2.1 mm × 12.5 mm Zorbax 5 μm 300SB-C3
guard column housed in a column oven set at 40 °C. The solvent
system used consisted of A: 0.1% formic acid in ultrahigh-purity water
(Millipore) and B: 0.1% formic acid in methanol (LC–MS grade,
Chromasolve). Chromatography was started in 90% A and 10% B at a flow
rate of 1.0 mL/min. A linear gradient from 10% B to 80% B was applied
over 35 s. Elution then proceeded isocratically at 95% B for 40 s
followed by equilibration under initial conditions for further 15
s. Protein intact mass was determined using a 6530 ESI-QTOF mass spectrometer
(Agilent Technologies Inc. USA). The ion source was operated with
the capillary voltage at 4000 V, nebulizer pressure at 60 psig, drying
gas at 350 °C, and drying gas flow rate at 12 L/min. The instrument
ion optic voltages were as follows: fragmentor 250 V, skimmer 60 V,
and octopole RF 250 V.

LC-MS data were acquired using an Agilent 6530 ESI-QTOF mass spectrometer with an Agilent 1290 UHPLC system. Using a flow rate of 0.3 ml/min on an RP C18 column (Phenomenex Kinetex 2.6 μm, 2.1 mm × 100 mm), acetonitrile/H₂O containing 0.1% formic acid (10%/90%) was held for 1 min, followed by a linear gradient of acetonitrile and H₂O (containing 0.1% formic acid) from (10%/90%) to (100%/0%) in 11 min, and finally, held for 2 min at acetonitrile/H₂O (100%/0%). Full scan mass spectra (m/z 100–1700) were measured in positive ESI mode. The mass spectrometer was operated with the following parameters: nebulizer pressure, 2.76 bar; dry gas flow, 10.0 L/min; dry gas temperature, 325°C; capillary, 3.5 kV; scan rate, 1 Hz. Two internal calibration compounds, purine and hexakis (1 hr, 1 hr, 3H-tetrafluoropropoxy) phosphazine, were used at low concentration throughout the acquisition. Targeted MS/MS data were acquired using two collision energies: 10 and 20 eV. IAA standard was purchased from Research Products International Corp (IL, USA).