|Unintended behavior when performing CE optimization with more than 10 product ions for a precursor||Chris Ashwood||2023-03-10|
OS/Skyline version: Windows 10, Skyline-daily 184.108.40.2065
Mode: Small molecule data
I hope you're all doing well. I am performing QQQ CE optimization for a single compound per run, with 14 product ions and 10 collision energies. As it currently stands, I have to modify the transition list after importing into Thermo software (Xcalibur 4.6 on an Altis Plus) as the export results in more than 10 rows with the same compound name and precursor m/z which causes an error. My understanding of the cause of the problem is below.
As Thermo method editor is limited to only a maximum of 10 rows with the same compound name and precursor m/z, Skyline appends a .2 to the end of transitions when there are more than ten. E.g. When I have 14 product ions and am performing steps in collision energy, there are ten product ions listed as "moleculeName(-charge).4", and four listed as "moleculeName(-charge).4.2". This behavior is expected and works fine.
The middle CE for a given molecule and product ion is just "moleculeName(-charge)" and, as I have 14 product ions for that name, there are ten product ions listed as "moleculeName(-charge)", and four listed as "moleculeName(-charge).2". This naming convention causes a problem as the second step in collision energy optimization uses the same suffix. E.g ten product ions listed as "moleculeName(-charge).2" and 4 product ions listed as "moleculeName(-charge).2.2". As a result, I then have a total of 16 compounds named "moleculeName(-charge).2", which is 6 more than allowed by the vendor software. In addition, it's confusing as I have to work out which of the "moleculeName(-charge).2" are from step 0 or step 2 in the CE optimization.
I currently append a B to the end of 4 of the compound names, but I'm not sure if Skyline can then read those files correctly. One possible solution could be changing the naming convention for CE optimization to add a 0 for the middle step in CE optimization, giving 10x "moleculeName(-charge).2" and 4x "moleculeName(-charge).0.2" in the example I'm encountering above instead of 16x "moleculeName(-charge).2". This should prevent any collisions in the naming as the transitions will then uniquely named.
Please find attached an example of the transition list that gets exported from Skyline, note the middle step for the last four product ions have the .2 suffix.