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iRT alignment issue when importing SWATH runs

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iRT alignment issue when importing SWATH runs laura declerck  2018-01-04 08:32
 
Hi,

Some time ago Skyline made it possible for me to build a library out of multiple searches on the same raw data files (Issue #501, "Improve BlibBuild handling of multiple searches on the same raw data files").
I needed this feature, because to annotate the iRT peptides in my data I needed different search parameters (Enzyme=Trypsine) than when I annotate histone peptides (Enzyme= ArgC, because all Lysine residues are blocked with a PTM or a chemical derivatisation by propionylation).

In the Skyline peptide and transition settings I chose the parameters that fit my histone peptides. This results in the fact that I am not able to retain iRT as a target, because I use 1) ArgC as an enzyme and 2) Propionylation (N-term) as a fixed modification.

When i want to import my SWATH results, iRT needs to be in my target list to be able to perform iRT alignment. However, to able to have iRT in my target list I need to change my settings in Skyline to 1) Trypsin as an enzyme and 2) no fixed modification on the N-termini. When I do this I lose a lot of my histone precursors and transitions, because they do not fit the settings anymore.

Is there a way that I can perform iRT alignment without loosing my targetlist? Would it for example be possible to use iRT alignment in the background, without putting it in the target list?

You can find the .sky file on this dropboxlink: https://www.dropbox.com/sh/pdyssp7hhdsndit/AACSrbExC0cT1fk1088gHYe2a?dl=0

Thanks a lot in advance for the help.

Kind regards,
Laura
 
 
Nick Shulman responded:  2018-01-04 14:08
I am not sure that I understand your question. I think I need to see your spectral library and iRT database.

In Skyline, you can use the menu item:
File > Share > (Complete) to create a .sky.zip file which contains your Skyline document and supporting files including the iRT database and spectral libraries.

If that .zip file is less than 50MB you can attach it to this support request here, or you can upload it to:
https://skyline.ms/files.url
(DropBox works too).

If your protein already has the correct peptides underneath it, and you want to prevent Skyline from automatically changing which peptides are there based on your enzyme or modifications, then you should turn off "auto manage children".
To turn off "auto manage children" for a Protein, right-click on the protein in the Targets tree and choose "Pick Children". Then, click on the magic wand icon so that the magic wand changes so that it has a red circle with a line over it.
When the magic wand has the red line through it, that means "Auto manage children" is off for that particular protein.

Skyline supports two different types of things at the top level in the Targets tree. One is a Protein, which has an amino acid sequence, and all of the children of the Protein are subsequences of the protein sequence. The other thing is a "Peptide List" which does not have a protein sequence, and can have arbitrary peptides under it.
Usually the iRT peptides are in a Peptide List, since, usually, iRT peptides do not come from the same protein. There are many ways to get a Peptide List into a protein. One is to choose "Edit > Insert > Peptides".