• Sign In
  1. Requests

Metabolites precursors: How to filter multiple hits when only 1 retention time detected.

support

View Request

view list print
Metabolites precursors: How to filter multiple hits when only 1 retention time detected. waltteri hosia22589  2024-10-15 06:23
 

Hello, back to Skyline after some time. Now testing metabolites precursor only HR workflow -with glycans, but question applies to metbolites in general I believe. Is there a way to filter so that skyline would choose only the nearest by RT in transition list as a hit, and discard other isobaric hits in transition list, and results -when there is only one retention time present?
With metbolites, and glycans in particular, isobaric structures are separated by retention time. Skyline picks all isobaric close-in-retention time compounds as hits despite they share the same RT/EIC. Restiricting retention time too tight dosen't work because retention times in transition list are predictions and because they can wobble a bit in hilic. In the attached screenshot Skyline marks all isobaric glycans to be there, G0, A2 and A1B, showing same EIC three times. The glycan nearest to predicted RT, A1B, is the only one actually there.
 
 
Brian Pratt responded:  2024-10-17 09:37

The glycan nearest to predicted RT, A1B, is the only one actually there.

The problem with this approach would be that the RT shift could just as easily cause Skyline to select the wrong glycan depending on the size and direction of the shift. Have you investigated iRT as a way to deal with chromatographic shifts? Also ion mobility separation is very useful in these cases if you have that equipment.

Thanks for using the Skyline support board,

Brian Pratt
 
waltteri hosia22589 responded:  2024-10-21 06:41

Thanks for reply. Yes, retention time based ID's for isobaric compounds can go wrong. I looked at iRT documentation, wonder how it would work for non-peptide compounds and hilic?
I think for Hi-Res experiments it would still not be bad if there was a user choice (on/off) button for one RT= one target type of results view. In my mind its not only getting retention time right but also results simplicity and logic. That when accurate mass data has one distinict EIC peak in certain RT window it cannot belong to two or more compunds which are known to differ chromatographically.
I am sure there could be better wording but something like this: "If two or more Hi-Res isobaric targets with overlapping retention time windows match one, same, RT peak in data, only the target with nearest RT to the data is assigned in the results"
 
Brian Pratt responded:  2024-10-21 08:56

Have a look at this:
https://skyline.ms/announcements/home/support/download.view?entityId=9c9fac12-b76c-103b-a2f7-22f5355603be&name=SmallMoleculeiRT-22_2.pdf

iRT really is the more general and robust approach. And, of course, ion mobility is extremely useful in these cases, if you have that gear available.