Exporting polarity-switching QQQ method from Skyline to Agilent-6495C apickard  2021-11-09 13:41
 

Hi,

We have recently begun using skyline to process our metabolomics data. We've had a lot of success trying this on Agilent GCMS, Agilent LC-QTOF and both Agilent and Sciex QQQ data files. We believe it will really speed up our data processing workflow once we get the hang of it. The "Synchronize Integration" option for integrations in Skyline Daily is a tool we have been looking for for a while and it has worked great for us so far. Today we tried to export transitions we have from a polarity-switching QQQ Skyline method into the Agilent 6495C using Skyline 21.1.0.278. We've noticed a few issues.

  1. The Agilent 6495C can go down to a dwell time of 0.5ms (which we are currently running), however, skyline only allows integers in the Dwell time (ms) field when exporting a method. We changed it to 1 in order to export the method without an error, but if it is possible to allow lower dwell times in a future release, that would be helpful.

  2. If we export and choose Polarity = "All", it is exporting our entire list of transitions (that are designated as either [M-H] or [M+H] adducts in Skyline), but it is changing all of them to positive polarity while keeping the m/z values for positive and negative MRMs. Since this method has polarity-switching, we would have to individually search through the transitions and manually change those that are [M-H] back to negative polarity. If we choose Polarity = "Separated by polarity", it appropriately splits the transitions into a positive and negative method with the correct transitions and polarity in each, so we know that Skyline is able to decide the correct polarity based on the adduct. We only have this issue when trying to have both polarities in one method.

  3. When we tried to export the method as Method type = "Scheduled", the only options are to use the average retention time, or use the retention times from a certain replicate. We were wondering if there was a way to instead use the defined explicit retention time for the compound instead of basing the RT on files that are open, or if this could be considered as a feature in a future release. This is especially helpful when we are using Skyline to process hundreds of MRMs and determining the compound RTs out of many different data files with different standard mixes (where some will include the compound and some will not).

Thanks for your help,
Amanda
 
 
Brian Pratt responded:  2021-11-10 13:32

Hi Amanda,

Thanks for the report.

For points one and two, can you supply the template file you're using to generate the method? With that I should be able to work with you toward a solution. Also your Skyline document (use File>Share>Complete) would be handy for clearest communication.

To the third point, I'll add an item to our development ideas list.

Thanks for using the Skyline support board,

Brian Pratt
 
apickard responded:  2021-11-10 15:18

Hi Brian,

Thanks for your help with this. Attached is my Skyline document as well as the template method (I zipped the .m file we are using in order to share). I also included the output method we are getting when trying to export all polarities (Test Method.m). Please let me know if you need any more information. We appreciate your help and all of the work you all do with Skyline.

Best,
Amanda