• Sign In
  1. Requests

How to manually specify masses for which to extract chromatograms?

support

View Request

view list print
How to manually specify masses for which to extract chromatograms? clindgren  2024-12-12 14:59
 

Hello,

I have an mzML file of unscheduled PRM data where I was continuously targeting some low abundance peptides. I've been searching the data with Fragpipe and then using the Skyline document generated by Fragpipe to look at the chromatograms for the targets. There are a few of the targets that Fragpipe fails to find, but I'd still like to extract chromatograms for their precursor and product m/z values to try and diagnose what's going on.

However, I can't figure out how to manually specify a peptide and its charge state, and then have Skyline extract chromatograms for the m/z values of its precursors and products. I've only been able to extract chromatograms for peptides that were successfully identified in the Fragpipe search.

I've tried using Skyline's built-in search with both MS Amanda and the built-in version of Fragpipe, but they also don't find the same targets, so I'm still not able to extract the chromatograms.

Is there a way to load an mzML file into Skyline, specify a list of peptides and charges, and then extract chromatograms for those targets from the mzML file, regardless of any associated search results?

Sorry if this is already answered somewhere--I've been reading through the tutorials and other forum posts and I keep thinking I've found the answer, but it's always not quite what I'm looking for.

Thanks,

Caleb
 
 
Nick Shulman responded:  2024-12-12 15:06
You can add a list of peptides to your document using the "Edit > Insert > Peptides" menu item.

If you need to also specify charges on those peptides there is a trick that works if you copy the correct text to the clipboard and use the "Edit > Paste" menu item.
If you wanted to insert a charge 2 peptide with the sequence "ELVIS" and a charge 3 peptide with the sequence "LIVES" then you could copy the following text to the clipboard and paste it into Skyline.
ELVIS++
LIVES+++

After you have inserted your new peptides into the document you can tell Skyline to extract chromatograms again using either the "Reimport" button at "Edit > Manage Results" or importing from new files with the "File > Import > Results" menu item.
-- Nick
 
Brian Pratt responded:  2024-12-12 15:07

Hi Caleb,

This tutorial
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_method_edit

shows a few different ways to get targets into Skyline, you probably want to concentrate on this section:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_method_edit&show=html

Thanks for using the Skyline support board,

Brian Pratt
 
clindgren responded:  2024-12-12 16:05

Thank you both! The "Edit > Insert > Peptides", then manually selecting the desired charges, and then using the "Edit > Manage Results > Reimport" button did the trick for me. The Reimport button was the key detail I hadn't been able to find before.

I did also try the "Edit > Paste" option with the appropriate number of "+" symbols appended to each peptide. When I did this, I pasted in a list of all my targets, both those that had been found and those that had been missed. After pasting, it came up with a message box listing all of the targets I was pasting that weren't already in the document, saying that they didn't pass the document's filter criteria and asking if I wanted to filter them out. This message box did give me an option to add them anyways, but when I selected that option they still weren't added, and it only added the targets I was pasting in that were already elsewhere in the document. Not sure if this is a bug, but it's not an issue in my case because I only have a small number of peptides, so I can just add them the other way and then go through and select the charge states manually.

Thanks for the help and quick response!
 
Nick Shulman responded:  2024-12-12 16:31
Caleb,

If you send us your Skyline document and the text that you were trying to paste, I can try to figure out what happened with the weird pasting behavior.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including spectral libraries and extracted chromatograms.

Files which are less than 50MB can be attached to these support requests.
You can always upload larger files here:
https://skyline.ms/files.url

I would certainly expect the peptides to be inserted if you chose "Keep" when Skyline told you that some peptides did not meet the filter criteria. Are you sure that these peptides were not just inserted in a weird place in the document? You can use the "Edit > Find" menu item to search for a peptide, or you could use the Document Grid.
There is information about how to use the Document Grid in the Custom Reports tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports
-- Nick
 
clindgren responded:  2024-12-12 21:31
I used the find menu to check that the peptides weren't added somewhere else, and I didn't see them. Weirdly enough though, of the ~45 peptides I tried to paste in, all of which did not exist previously in the document, 2 did actually add successfully--FSAFFGNAPQFTIPGR+++ and GAYITTVGDYVGNYK++. I don't see anything about these two that would distinguish them from the rest, although it is interesting to note that they're right next to each other in the list I'm pasting.

I uploaded the .zip file to the link you gave, it's called fragpipe_skylib.sky.zip and is about 67 MB. I've attached a text file of the targets I'm trying to paste in, as well as a screenshot of the message box about the peptides not matching the filter criteria. This is the box where I clicked the "Keep" button but the peptides were not kept.

Thanks!
 
clindgren responded:  2024-12-12 22:17
One other quirk I've noticed: With the "Edit > Insert > Peptides" followed by manual charge picking followed by "Edit > Manage Results > Reimport" method, after the reimport some of the added targets have chromatograms extracted for both their precursors and products, whereas others only have chromatograms extracted for their precursors. I've attached screenshots of both cases. How do I get Skyline to extract chromatograms for the products as well as the precursors for all the peptides I've added?

If you want to recreate the situation, I ran into it starting from the same Skyline document and missed target list as from my previous message. I've also attached the same target list to this message, but without the "+" signs after the peptides, so that you have a copy of the target list that's correctly formatted for the "Edit > Insert > Peptides" box.
 
Nick Shulman responded:  2024-12-12 22:40
If you do not get MS2 chromatograms for a particular precursor after reimporting that usually means that there really were no MS2 spectra whose isolation window matched the m/z of the precursor in the Skyline document.
I see that at "Settings > Transition Settings > Full Scan" the Acquisition Method is "DIA" and the Isolation Scheme is "Results Only".

I would have to see your .raw or .mzML file to know for sure, but I assume that there were no MS2 spectra where the isolation window contained the m/z 579.2645 in your screenshot.

You can use the program "SeeMS.exe" (which comes with ProteoWizard) to look at the spectra in your raw or .mzML file and see whether there are any spectra with the necessary isolation window.
-- Nick