Help getting mods to show up on peptides

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Help getting mods to show up on peptides Theresa  2017-08-14 14:22
 
Hi,

I have a list of peptides of interest. I've put CAM as a fixed mod and variable mods including S/T/Y, oxidation, acetylated N', and N/Q deamidation. Only the CAM is showing up on the peptides and the exported transition lists. I've made sure that variable box is checked for all variable mods. I'm not sure what else to try.

Thanks for any help you can offer,
Theresa
 
 
Brendan MacLean responded:  2017-08-14 14:32
Hi Theresa,
It is not clear from your description or screenshots where exactly the modifications do not "show up". They seem to be showing up just fine in your settings.

I assume you must mean that they are not showing up on targets you are adding to the Targets view? How are you adding them? If you add them by File > Import > FASTA or Edit > Insert > FASTA or just Edit > Paste some FASTA format text from the clipboard, then you should get peptides with variable modifications, unless you are requiring Skyline to pick only peptides which match a spectral library and that spectral library does not actually contain and peptide IDs with those modifications.

Anyway, it is going to take a bit more understanding of both your other settings and how you are adding targets to your document to know why you are not seeing variable modifications.

You can also post your document to:

http://skyline.ms/files.url

For us to have a closer look.

Thanks for posting the screenshots with your request.

--Brendan
 
Brendan MacLean responded:  2017-08-14 14:33
If you post your Skyline document, use File > Share - Complete to create a .sky.zip file containing all the component parts.
 
Theresa responded:  2017-08-15 08:22
Hi Brendan,

I have used the both Edit->Insert->Peptide (attached) and "Import peptide list" from the home screen when opening SLDaily. I am trying to export a transition list which includes 2,3,4 charge states, fixed CAM, and the variable mods you'll see. Only the fixed CAM shows up on the export list (or in the table of peptides). I'm sure I'm doing something wrong but I've run out of ideas.

Update1: I tried to add the zipped file and it is larger than the allowable file size. (Because of the human library?)
Update2: It looks like it uploaded to the link you shared.

Thanks for your help,
Theresa
 
Theresa responded:  2017-08-16 19:19
Hi Skyline team,

I'm just checking in to see if you've had a chance to look at my file. I am still unable to get the variable mods to appear in the transition list.

Thanks for your help,
Theresa Russell
 
Nick Shulman responded:  2017-08-16 22:16
One way to get a modified peptide into your document is to right-click on one of the peptides in the Targets tree and choose the menu item "Modify".

If you want Skyline to add all possible modified peptides for all of the proteins in your document, you can go to:
Edit > Refine > Advanced
and choose "Auto select all: peptides".
Skyline will add 130 thousand peptides to your document.

If you want to add some modified peptides to a particular protein, you can hover the mouse over the area just to the right of a protein name in the Targets tree and click on the inverted triangle that appears. It takes several seconds for the popup pick list to appear because there are so many choices, but you will then be able to check the checkboxes next to the modified peptides that you want, or you can click the magic wand button to select them all.
 
Theresa responded:  2017-08-17 07:03
Hi Nick,

Thank you for taking to the time to provide such a detailed answer. I'm grateful and I'll explore the options you mentioned.

I see now that I'm trying to use Skyline in a way that it is not designed to be used. I was thinking that by having particular variable mods checked, as I do, the software would apply those possibilities to the peptides in my document and include those specific possibilities in the exported transition list. i.e - the peptide with transitions +/- phosphorylation. I had hoped to import the transition list and increase the chances of detecting the modified peptides. I was hoping that an automated process was available.

Thanks again for your help and have a great day!
Theresa
 
Brendan MacLean responded:  2017-08-17 12:19
Hi Theresa,
We spoke by WebEx, and I hope it became clear that Skyline is definitely designed with the intent of supporting what you are trying to achieve, but that you had gotten somewhat off the track we have designed and hoped you would use.

In the end, we came up with an approach that should get you most of the way to what you were hoping to achieve, with some extra manual work still required for your non-tryptic peptides.

The high-level workflow can be summarized as:
1. Create a broad target list covering your proteins of interest.
2. Use Edit > Refine > Accept Peptides to narrow the list to only the peptides you are interested in and all of their modified forms.

In our first attempt on the phone call we did this without any spectral library filtering and ended up with around 18,000 modified forms of your the 143 peptides out of your 160 desired that were fully-tryptic and not too long for your settings.

Then, because you mentioned the Global Proteome Machine (GPM) as the source of your information on what to monitor we started downloading GPM libraries directly from the FTP site (ftp://ftp.thegpm.org/projects/xhunter/libs/eukaryotes/lib/human_chromosomes/), which we, unfortunately, had to do 1 chromosome at a time. (I got through 3, 5, 11, 15, 16 and 20.) With those plus the NIST Human library I got 428 modified peptide forms with 549 precursors. After applying Edit > Refine > Accept Peptides with your 160 peptide list, I was left with 102 modified peptide forms with 131 precursors.

See attached PowerPoint slides for an overview of the steps we took.

Thanks for the quick WebEx this morning.

--Brendan
 
Theresa responded:  2017-08-26 07:49
Hi Brendan,

Thank you for your help in getting my project back on track. I was able to include all of my peptides of interest (POI) with their modifications, many of which Skyline identified automatically and then manual entry for the rest. An important part of the learning process for me was appreciating that the spectral library you choose makes a big difference. For example, many of my POIs can be found in online searches of the Global Proteome Database and/or PeptideAtlas but are not included in the downloadable version of their spectral library files.

Having struggled to do much of my MRM workflow setup manually, I now have a deep appreciation for the power of Skyline. I'm so impressed with the customer service provided by your team and I really can't thank you enough for taking the time to help me. My next project will come together much more quickly!

Gratefully,
Theresa Russell
 
Brendan MacLean responded:  2017-08-26 08:58
Great news! Thanks for the update. Good luck with your research.