To begin, TIMS-TOF MS/MS Method development, open the TIMS control application. Under MS settings, set the scan beginning and ending values to 50 mass to charge ratio and 1, 800 mass to charge ratio, respectively. Select the ion polarity mode as positive and the scan mode as parallel, accumulation serial fragmentation or passive.
Next, move to the tabs under source and set the end plate offset to 500 volts, capillary to 4, 500 volts, nebulizer to 3.0 bar, dry gas to 10.0 liters per minute and dry temp to 200 degrees celsius. In the syringe pump settings, assign the syringe as Hamilton one milliliter. Enable the option active and set the flow rate to 80.0 microliters per hour.
In the MS/MS tab, under precursor ions, set the number of passive ramps to eight, minimum charge to one and maximum charge to five. In scheduling settings, enable the precursor repetitions and under active exclusion, check the box to enable and set the release to 0.40 minutes after. Do not adjust the collision energy settings and isolation with settings.
Then click to enable the TIMS stepping box. To perform calibration for both mass to charge ratio and mobility domains, under the mass to charge ratio calibration settings, select one of the preloaded tuning mix profiles in the reference list box. To calibrate, load the syringe for the TOF with a tuning mix solution.
In the calibration mode, on the right side of the window, select between linear, quadratic, and enhanced quadratic to achieve the best score
In the dropdown menu, select save as to generate a new MS method file.
