ESI Experimental Section - sample
ESI conditions when using the Agilent 6540 QTOF
Electrospray ionization was performed on an Agilent 6540 QTOF mass spectrometer (Agilent, Santa Clara, CA) coupled with an Agilent 1200 Series HPLC system. The instrument was tuned and calibrated using the Agilent ESI tune mix( ). Resolution of 45,000 (FWHM) at mass 922 was achieved with 4 GHz digitzing rate. The mass spectrometer was scanned from 100 to 2000 daltons. 1 μL of sample was injected via the Agilent .... Autosampler. In the flow injection mode, the divert valve is set to column 2 position, while column 1 position was reserved for the LC column path. Concentration of 10 to 100 ppm (10-100 ng/ul) is typical when using flow injection.
Tipical LC conditions:
LC column: C18 XDB 1.8 um, 4.6/150 mm
H2O/ACN 95/5 to H2O/ACN 5/95 in 10 min
ESI conditions when using the TSQ700
Samples were introduced into the electrospray interface through an untreated fused-silica capillary with a 50 μm i.d. and 190 μm o.d. A myoglobin and MRFA mixture was used for tuning and routine calibration of the instrument. The mixture was made in the following manner: stock solutions of myoglobin and MRFA were prepared by dissolving 0.9 mg of horse skeletal muscle myoglobin in 1 ml of a solution of 50:50 methanol: water containing 1% acetic acid, and 0.6 mg of MRFA was dissolved in 1 ml of a solution of 50:50 methanol: water containing 1% acetic acid. The calibration and tuning standard was made from the two stock solutions: 50 μL of myoglobin and 12 μL of MRFA were diluted with 538 μL of a 50:50 methanol: water solution in a polypropylene vial. The tuning mixture was infused into the mass spectrometer at a rate of 3 μL/min on a Harvard Apparatus (model 22, South Natick, MA) syringe pump.