Unintended behavior when performing CE optimization with more than 10 product ions for a precursor

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Unintended behavior when performing CE optimization with more than 10 product ions for a precursor Chris Ashwood  2023-03-10 08:46
 

OS/Skyline version: Windows 10, Skyline-daily 22.2.1.425

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.

Cheers,
Chris

 
 
Nick Shulman responded:  2023-03-10 13:13
Skyline does not pay attention to those compound names when you are importing chromatograms from SRM data, so you do not need to worry about confusing Skyline with your renaming.
Skyline matches chromatograms to transitions by looking at the Q1 and Q3 m/z values (as well as the time range of the chromatogram if the molecule has an Explicit Retention Time).

It sounds like the ambiguity comes from the fact that Skyline does not include the charge in the compound name if the charge is 1, and that Skyline does not include the CE optimization step number in the compound name if the optimization step is 0. The fix is probably that Skyline should either always include the charge or always include the optimization step if there is any optimization going on.

Can you send us an example of a Skyline document where the compound names in the exported transition list are not working right?
-- Nick
 
Chris Ashwood responded:  2023-03-10 15:39
Hi Nick,

That's great news, thank you.

The charge is included for -1, it shows up as --1. From what I have seen, they always include the charge state. I am working in negative mode, but I'm not sure if that matters for this.

The attached transition list above goes with the Skyline document I have attached here.

Thank you,
Chris