How to create a SONAR calibration file after acquisition - WKB202040

OBJECTIVE or GOAL

Manually create a SONAR calibration file (_sonar.inf) for data reading and processing in third-party software such as Skyline. 

ENVIRONMENT

• Windows 7 Professional 64 bit SP1 

• Windows 10 Enterprise LTSB 2016 

• SYNAPT XS 

• Xevo G2-XS 

• MassLynx 4.1 (Xevo G2-XS only) 

• MassLynx 4.2 

• Apex3D (distributed with PLGS, Progenesis QI for proteomics, and DriftScope) 

• Skyline 

PROCEDURE

1. The quadrupole calibration of a SONAR acquisition is stored in the _sonar.inf file, which resides in the .raw sample folder. Shown in the figure below are the location of the calibration file (left) and its content (read with a text editor such as Notepad; right).

2. Create a copy of the provided _sonar.inf template (click link to download attachments) and manually update the first four lines of the [SONAR#1] section. The information can be obtained from the MassLynx method editor that was used to create the SONAR acquisition method.
3. Peak detect the data using Apex3D (distributed with PLGS, Progenesis QI for proteomics, and DriftScope; executed from a command line or batch file) using one the example sets of arguments shown below. An example batch file (SONARcalibration.bat) is also provided in the attachments section of this article. The paths and arguments highlighted must be updated using a text editor. After the changes are made, save the file and execute it by double-clicking.

…\lib\Apex3D64.exe -pRawDirName …\filename.raw -outputDirName …\outputfolder -outputUserDirName …\outputfolder -lockMassZ2 785.8426 -lockmassToleranceAMU 0.25 -writeBinary 0 -bCSVOutput 1 -leThresholdCounts xyz -startingRTMin xyz -endingRTMin xyz -function 1  

…\lib\Apex3D64.exe -pRawDirName …\filename.raw -outputDirName …\outputfolder -outputUserDirName …\outputfolder -lockMassZ1 556.2771 -lockmassToleranceAMU 0.25 -writeBinary 0 -bCSVOutput 1 -leThresholdCounts xyz -startingRTMin xyz -endingRTMin xyz -function 1

Update the paths and specify names and/or data file specific values for the labeled arguments. 

4. This process creates a filename_Apex3DIons.csv file containing peak detected data over the specified retention range above the detection threshold. 

5. Open the file in Excel and create a scatter plot with m/z on the x axis and mobility, atDrift1Start, and atDriftStop on the y axis. Mobility represents the center of the isolation window, and atDrift1Start and atDriftStop the edges, respectively. An example is shown below. 

6. Add linear trend lines and display the equations, as shown, on the chart. 

7. Calculate for at least two m/z x-axis data points, ideally the start and the end of the SONAR quadrupole scan window, m/z 400 and m/z 900 in this example, the corresponding bin y-axis values for the three equations. In this example, m/z 400 would equate to 0.4014*400 - 159.18 = 1.37 for the calibration line representing the center of the quadrupole isolation window. Including the information from the other two calibration lines, this creates the following line for the [SONAR#1] section: Calibration=400,-5.83,1.37,9.59  

8. Update the last calibration information of [SONAR#1] section, providing a result as shown below, and save the _sonar.inf file.  

9. Distribute/copy the created _sonar.inf to all the .raw data file folders of the complete experiment. Calibrating individual files is not required.  

ADDITIONAL INFORMATION

NOTE : The described manual procedure in intended for SONAR acquisitions with the same LE and EE scan range and scan time.

Glossary of commands used in the batch file: