New to SkyLine- In silico

New to SkyLine- In silico New to Skyline  2022-11-29

Hi All,

I am new to skyline. I am doing a research project in measuring a particular hormone. Is there a 101 document on how to use the Software, I have been through the tutorial (targeted method editing) but unable to replicate it for my project. We have the water Xevo. I wanted to do an in-silico for my peptide but dont know how to conduct it. Thank you for any support.

Kind regards,

Nick Shulman responded:  2022-11-29
When you add a Protein to your Skyline document, Skyline always performs an in-silico digestion on the protein sequence and the peptides also appear in the Targets tree.

There are a couple of different ways that you can add a protein to your Skyline document.
One way is by using the menu item:
Edit > Insert > Proteins
In the Insert Protein List dialog you just need to fill in something in the "Name" column and also put the entire protein sequence in the "Sequence" column.

Skyline will add the new protein to the Targets tree. Skyline will also perform a digestion on the protein sequence and add the peptides that match your filter criteria. The filter criteria that Skyline uses is specified on the "Digestion" and "Filter" tabs of the "Settings > Peptide Settings" dialog.
If you do not see any peptides in the Targets tree under your new protein, you might be able to choose some by right-clicking on the protein in the Targets tree and choosing "Pick Children"

A different way to add proteins to the document is by using the menu item:
File > Import > FASTA

I hope this helps.
Let us know if you get stuck anywhere.
-- Nick
New to Skyline responded:  2022-11-29
Dear Nick,

Perfect- Thank you. My knowledge of this software is at its infancy. Is the first option (edit>insert>protein) the digest using tryptic at cleavage site KR or other. This did not create a library match spectrum graph, why is that?

General Question: [relating to the method editing document]

1) The spectral library creation- Is this to create and gather all the possible surrogate proteins & peptide available for an overall protein class?
1.1) Do you need to search & download a FASTA file for the protein of interest to be used by skyline or is this automatically retrieved by skyline e.g. Yeast (Atlas) stated on document

2) the second option of File> Import >FASTA, Ive tried this process (with downloading the FASTA file from NIH) but once entered, the target section remains blank (the first process you stated worked)

3) Checking peptide uniqueness- How do you add a background Proteome and how would i go about in looking for this?

4) what does "Trypsin [KR | P]" indicate? is it the trypsin digest occurring at the Lysine and Arginine terminal only?

Thank you again.

Kind regards.
Nick Shulman responded:  2022-11-29
Edit > Insert > Protein
would use whatever enzyme you have selected at "Settings > Peptide Settings > Digestion". That defaults to being Trypsin.

You would only see something in the Library Match window if you had a spectral library. You can also right-click on the Library Match window and choose "Prosit" which means that instead of Skyline showing you a spectrum from your spectrum library, Skyline instead will ask a server in Germany to use its machine learning algorithm to predict the fragmentation pattern of the currently selected precursor.
(If you still don't see anything in the Library Match window after selecting "Prosit", you probably don't have any peptide or precursor in your document. You should send me your Skyline document (see answer #2 below))

1) The spectral library tells Skyline which fragment ions are likely to be detected for any given peptide.

1.1) Skyline does not know how to download FASTA files from anywhere, so you should download it yourself.

2) Can you send us your FASTA file and your Skyline document?
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files such as spectral libraries
If that .zip file and your FASTA file are less than 50MB you can attach them to this support request.
You can upload larger files here:

There are lots of potential reasons that Skyline might decide not to add any of the proteins from your FASTA file to your document and after I see your FASTA file and your I will be able to tell you what is going wrong.

3) You create a background proteome by going to "Settings > Peptide Settings > Digestion" and choosing "<Add...>" from the Background proteome dropdown. If you are just starting out with Skyline I would discourage you from choosing anything other than "None" in the "Enforce peptide uniqueness" since Skyline automatically removing non-unique peptides from your document sometimes results in confusing behavior.

4) "[KR | P]" indicates that the enzyme cleaves at Lysine or Arginine unless that amino acid is followed by a Proline. If you want to see the details of the currently selected enzyme you can go to:
Settings > Peptide Settings > Digestion
and choose "<Edit current...>" from the Enzyme dropdown and Skyline will show you a dialog with all of the details about the currently selected enzyme.

-- Nick
New to Skyline responded:  2022-11-30
Hi Nick,

Thank you.

Prosit- I have gone to peptide setting< Library<Build< Data source, and selected 'Prosit' but it requires to be configured, how do I come about in doing this? I do not see anything in the library match window.

I have attached the shared the skyline document for the sequences.
Nick Shulman responded:  2022-11-30
In order to get Prosit to work in Skyline it might be necessary to go to:
Tools > Options > Prosit
and make sure that something is selected in all of the dropdowns.

I kind of think I have seen the behavior that sometimes when you first install Skyline sometimes the "Intensity Model" or "iRT model" starts out blank and you need to go into the Tools Options dialog to choose something. This might be a bug in Skyline.

-- Nick
New to Skyline responded:  2022-11-30
I am still unable to see a spectral library and have an error code. I have attached
Nick Shulman responded:  2022-11-30
I do not know how to figure out what is going wrong with your use of Prosit. You probably do not need to worry about that now.
Is there anything else that you have questions about?
-- Nick
New to Skyline responded:  2022-12-01
Hi Nick,

> A was able to get the library match spectrum to open, but it states the spectrum information is unavailable.

> What are the process of using Skyline, after setting the sequences and surrogate sequences have been generated?
- Is there a way of indicating which digest peptide are unique vs generic.

> Do you have a step by step document in using Skyline/ query document

Kind regards
Nick Shulman responded:  2022-12-01
Your Prosit problem might be caused by your institution's firewall in between your computer and the Internet.
It might work for you if you put your computer on a different network such as your phone's hotspot.
There might be some useful information in this support request:
Brendan had a detailed answer on October 28th. Skyline communicates with the Prosit server over port 8500, and we have a theory that maybe some IT departments are blocking that outgoing port. We do not have any real evidence that this is happening, but if you ask your IT department to look into this they might be able to give you useful information about whether this is something that they can fix for you. Let us know if you learn anything that we could share with other Skyline users about this issue.

When it says "Spectrum information is unavailable" that either means that you do not have a spectral library, or, if you do have a spectral library, the library does not contain a spectrum for the currently selected peptide.

In Skyline, if you go to:
Settings > Peptide Settings > Library
and then press the "Edit List" and "Add" buttons, there are some links where you can download spectral libraries from PeptideAtlas, NIST, and GPM.
After you download the spectral library, you will probably have to uncompress it, and then you can press the Browse button on the "Edit Library" dialog to add the spectral library to your document.

If you are interested in unique peptides, then you should create a background proteome and choose something from the "Enforce peptide uniqueness" dropdown. (I realized I discouraged the use of the uniqueness dropdown before, but that was because it sounded like you did not have any peptides in your document, and I was worried that Skyline might remove all of the peptides from your document because none of them were unique)

For triple quadrupole machines like the Waters Xevo, the best tutorials are the "Targeted Method Editing" and "Targeted Method Refinement" tutorials:

There are also some webinar links on both of those pages.

I am not sure exactly what you mean by "query". If you are asking about how to get lists of numbers out of Skyline, you should take a look at the Custom Reports tutorial:

-- Nick
New to Skyline responded:  2022-12-05
Thank you, I have contacted our IT system and will update.

How do you download the spectral libraries at PeptideAtlas.
I have gone to< Human all (2022-01) < folder Biosequence Set FASTA format (598MB) [Is this the correct file).
This leads me to this page:
 edit library< peptideatlas< ISB< Human (phosphor)

Ive unzipped the folder but when I try and build the library the folder itself is empty.
New to Skyline responded:  2022-12-05
Thank you, I have contacted our IT system and will update.

How do you download the spectral libraries at PeptideAtlas.
I have gone to< Human all (2022-01) < folder Biosequence Set FASTA format (598MB) [Is this the correct file).
This leads me to this page:
 edit library< peptideatlas< ISB< Human (phosphor)

Ive unzipped the folder but when I try and build the library and importing it via skyline the folder itself is empty. The folder itself