For nanoflow workflows using the ZenoTOF 7600 system and the standard nanoflow interface, on-column calibration is required and can be incorporated automatically into the SCIEX OS software acquisition batch. Here, step-by-step instructions are provided to set up a custom calibration table using PepCalMix, which is a mixture of 20 stable isotope-labeled peptides that is used for instrument quality control experiments. The approach described here could be used for any custom standard desired for on-column nanoflow calibration.

To set up auto-calibration in a batch, click the blue Auto-Calibrate button at the top of the SCIEX OS software Batch screen to launch the Batch – Automatic Calibration Editor.  Note: if you are using a different LC system through contact closure, you will need to add the diverter valve to your hardware profile to make the Auto-Calibration button appear.In the Batch – Automatic Calibration Editor, click Edit to build a new ion reference table that corresponds to the sample used for calibration.

In the Ion Reference Table editor, click New to create a new table.

In the table, enter the name of each compound and its corresponding precursor m/z (Da), collision energy (CE), declustering potential (DP) and retention time. Then, select which compound will be used for MS/MS and provide the reference ion information in the MS/MS table. Enter a retention time tolerance with unit seconds.

Click the Save button and enter a name for the calibration table. The calibration table will be accessible for all future MS workflows. In this example, a method for PepCalMix was built that incorporated the TOF-MS/MS fragmentation of the 758.9015 m/z precursor, as indicated in the ion reference table built in the Ion Reference Table Editor window, shown above.

If using PepCalMix for your calibration, please refer to this Excel file, which contains the m/z values for TOF-MS and TOF-MS/MS analyses, CE for MS/MS analysis and DP values for all isotope-labeled peptides included in the mixture. These values can be pasted directly into the ion reference tables in the SCIEX OS software.

Once the ion reference table is built, complete the remaining fields in the Batch – Automatic Calibration Editor. Input the name of the ion reference table created, calibration frequency and the LC method to use for calibrant delivery. For the MS method, use TOF-MS and TOF-MS/MS methods that correspond to the samples used for calibration. Note that TOF-MS and TOF-MS/MS methods must be used specifically, as MRM^HR methods, for example, are not compatible with calibration. The LC settings indicated in the Batch – Automatic Calibration Editor should correspond to the vial in which the sample is located. Note that there is no option for injection volume. For M-class systems, as used in this example, the default injection volume is 1 µL for on-column calibration.  If using an LC system through contact closure, create a dummy diverter valve method and include in the batch, this allows you to use Auto-Cal with your LC system. Just remember to insert an extra LC run in your offline LC batch for every Cal run on the MS.

Click OK in the Batch – Automatic Calibration Editor to return to the SCIEX OS software Batch screen. To begin calibration, make sure the checkbox is selected next to the blue Auto Calibrate button. When submitting a sample, the calibration will commence and proceed with the frequency designated in the Batch – Automatic Calibration Editor. If the MS method is changed, a new calibration will automatically be performed. After submission, you will see your calibration runs as rows in the batch. Now, you are ready to perform on-column calibration for nanoflow experiments using the ZenoTOF 7600 system!

For information on automatic calibration when running microflow LC, please see the community post on Automatic calibration in minutes for microflow LC with the OptiFlow source on ZenoTOF 7600 system.

RUO-MKT-18-14611-A