How to set up a Tof MRM experiment - WKB5226

OBJECTIVE or GOAL

Create a Tof MRM method

ENVIRONMENT

• Xevo G2-XS
• Synapt G2-S
• Synapt G2-Si

PROCEDURE

1. To set up a Tof-MRM experiment, you must choose the Tof-MRM acquisition type in the MS method editor window.
2. In the Acquisition tab, specify the basic method parameters, including time length, source type, polarity, and Tof mode.

4. In the Tof MS tab, input parameters specific to the Tof analyzer, including mass range, scan time, and data format. The mass range should encompass all masses of interest. This is particularly applicable when you use RADAR or None for the MRM window (see step 5). 

5. The MRM tab contains parameters that are specific to the MRM experiment to be performed. MRMs can be scheduled manually by inputting start and end times, masses of precursors and fragment ions, collision energies, cone voltage, and a Target Enhancement mass, if used (this should be set to the daughter ion mass). MRM schedules can also be imported directly as a *.mrm file from a previous experiment or from an Excel spreadsheet. Other options on the MRM tab include toggling of RADAR, scan padding, and data stripping, if required (see descriptions below). 

Radar - If you want, you can schedule a full Tof MS spectrum scan to provide data on what else is present in the sample, outside of (in addition to) the MRM transitions that are the principal focus of the experiment. 

Scan Padding - If multiple, overlapping MRMs are scheduled such that the number of simultaneous MRMs changes as the run progresses, it is best to enable Scan Padding. This feature inserts dummy functions into regions where the number of consecutive MRMs drops, so that the amount of time spent looking for any given transition is consistent over the duration of the experiment. Accurate quantitation requires this setting.  

Data Stripping - By default, MassLynx stores data that covers the entire acquisition mass range specified in the Tof MS tab. This is the None option in the MRM window. This option is not recommended. For ease of viewing and to greatly reduce the file size, you can use data stripping to limit the Tof MS data that is written to disk. The Isotope Cluster option retains data within a window of -1 to +5 Da around the target fragment ion mass, while the Single Isotope option retains data within a window of -0.2 to +0.2 Da centered on the target fragment ion mass. 

6. To add an MRM transition to the method, select Add to open the MRM editor. Enter the compound name if desired. Enter the retention time window and the parent mass under Set Mass. You can enter more than one fragment ion under Fragments. Set a collision energy ramp by entering the low and high collision energy in the Trap (or, if present, Transfer) CE Ramp windows. If you need no collision energy ramp, enter the same collision energy in both windows. A good ramp for peptides is 10 - 45. Enter the cone voltage desired. For the Target Enhancement m/z, enter the m/z of the main daughter ion. When you are satisfied with the MRM transition, select Add, and then select Save to save the *.mrm file. This only saves the MRM transition to a file, not to the MS method. When all of the MRM transitions have been saved to a *.mrm file, then save the Tof MRM MS method (*.exp).

7. Be sure to sort the transitions before saving the method.

 

ADDITIONAL INFORMATION

The MS method reuses functions so that only a minimum number of functions are created. Although there may be ten MRM transitions in the method, there may be only three functions. To see which function each transition was acquired to, browse to the data file.raw folder and open the *.fdc file with Notepad or, better yet, Excel. This shows a list of the function and each transition. 

To open the .fdc file with Excel: Open Excel, select file open, and browse to the file. Select *.* for the file type, select Delimited, select Next, and then check all of the Delimiters except other. Select Next, and then select Finish.