How to manually calibrate CCS values for SYNAPT G2 HD data - WKB25249

OBJECTIVE or GOAL

Calculate collisional cross section values for SYNAPT G2 mobility data from drift bins so that CCS values can be imported into Progenesis QI for Proteomics.

On the SYNAPT G2-S/Si, this is relatively automated. You run CCS calibration in IntelliStart, and the mobility data acquired in MassLynx is then automatically reported as collisional cross section (CCS), rather than drift time. Progenesis QI picks up that CCS information when the .Raw data is imported. On the SYNAPT G2, the IntelliStart option for CCS calibration isn't available. But the demo lab has shown that it's possible following the steps below.

ENVIRONMENT

• SYNAPT G2

PROCEDURE

1. Create a calibration.
   1. Acquire polyalanine mobility calibration data using exactly the same instrument settings used for sample acquisition.
   2. Save the *.csv polyalanine reference file (polyalanine ref_cal.csv) in the DriftScope “cal” subfolder (if it isn't there already).
   3. Open the previously acquired polyalanine data in DriftScope.
   4. Click on the “Detect Peaks” button and use the generic settings (a spot is placed on every detected peak apex).
   5. Click on “Peak Detection” and select Calibrate Peaks > New Calibration. A new “CCS Calibration Editor” window opens.
   6. Click File > “Open reference file…” to load the polyalanine *.csv reference file.
   7. Click Peaks > "Auto select…” and set m/z tolerance = 0.1Da and Intensity Threshold = 0, to generate an ion mobility calibration curve for your acquisition settings.
   8. Click Calibrate > “Save calibration” to save the calibration you have just generated. The CCS Calibration Editor window closes automatically and the original DriftScope data window reopens.
2. Apply the calibration to sample data.
   1. Open your sample data in DriftScope and apply “Detect Peaks” in exactly the same way as for the polyalanine data.
   2. The appropriate “Minimum Intensity Threshold” value should be chosen for each individual data file (too high and very few peaks are detected, too low and the whole data set is covered in spots, including all the low level background ions!).
   3. Click Peak Detection, select Calibrate Peaks… > Apply Calibration…, and do a single left mouse click on the calibration file you previously generated (the name of the calibration file matches that of the original raw data file).
   4. Click Open – this applies the calibration file to the sample data that is open in DriftScope.
   5. To zoom in on a region of interest in DriftScope, click on the “Use Zoom / Combine Tool” button, and then hold down the left mouse button and draw around the required region.
   6. To view the calculated CCS values for your sample data, zoom in to a region of interest in DriftScope, hold down the Control (Ctrl) key on your keyboard and hover the mouse pointer over the apex spot that corresponds to the ion of interest.

After doing this, the CCS value persists in the data file, and if the data is imported into Progenesis QIp or UNIFI, the CCS values should also import.

ADDITIONAL INFORMATION

It's possible to automate the step where you copy the calibration file into the relevant .raw folders using a bat file containing the following commands ("." indicates the name of the raw file, and the file paths of the raw file and the mob_cal.csv must be changed to reflect the correct locations of your files):

@echo off
for  /D  %%a  in ("d:\test\test2\*.*") do xcopy  /y  /d  d:\test\mob_cal.csv "%%a\"