Display/integration error when using small molecule iRT calculators

Display/integration error when using small molecule iRT calculators Chris Ashwood  2022-08-04

Dear Skyline team,

I hope you are doing well. When I have set up the small molecule iRT calculator and begin integrating peaks using the "Show XXXX Score" display, I'm encountering a few issues after successful calibration:

  1. The windows are not updated from "Retention time" to "XXXX Score" for both the file-specific chromatogram window and the "Retention Times - Replicate Comparison" window. This appears to happen the first time I click on each molecule, but after I click back and forth, Skyline then displays the right window.
  2. The first time I integrate inside the file-specific chromatogram window when it is set to "XXXX Score", the display bugs out and the integration appears to be different from what I selected in the window file-specific chromatogram window. I think it is setting the iRT value used for integration as the retention time instead of the iRT score (e.g. integration from 1 to 10 score instead integrates 1 to 10 minutes in the retention time view).

The Skyline file used for this is attached. Happy to give more detail.


Nick Shulman responded:  2022-08-04
I am not sure I understand your question. Can you send us some screenshots?

A lot of these graph windows remember separately which retention time calculator they are using, so choosing a different calculator in one window is not necessarily going to change the other windows.

By default, the calculator for the "Retention Times Regression Score to Run" window is "Auto", which means that Skyline tries all of the different calculators and chooses the one which performs the best. In your small molecule document, Skyline seems to end up choosing the calculator "SSRCalc 3.0 (300 A)", which does not look like the right thing to be doing, because all of the points end up plotted in a vertical line. It looks like Skyline is not properly handling NaN ("not a number") when comparing the r values of different linear regressions.

-- Nick
Chris Ashwood responded:  2022-08-05
Hi Nick,

Sorry I wasn't clear. It's a little tough to explain and I was hoping the audit log would be more informative.

Please find attached a pdf with labelled screenshots, I hope that makes the error I'm encountering clearer.

Nick Shulman responded:  2022-08-07
I see what you are saying. When a new chromatogram window is activated, it first displays itself with the ordinary retention time numbers on the X-axis, and the next time it has to update itself, it switches to displaying the iRT calculator score, but, by that time, the location that has been zoomed to is completely inappropriate for the iRT scale.

By the way, the audit log only keeps track of changes to the document. Telling Skyline to show the iRT score on a graph does not actually change the document (i.e. it would not cause you to be prompted to save the document when you exit) so there will not be any entries in the audit log for an operation like that.

I will try to fix this chromatogram behavior.
-- Nick
Chris Ashwood responded:  2022-08-07
Hi Nick,

Excellent, sorry for the vague report! What you’re saying is the problem I’m encountering.

Ah, that’s interesting. Thanks for letting me know. I didn’t realize iRT doesn’t change the document.

Thank you, hopefully it’s an easy fix!