Determine sequence coverage for proteins

support
Determine sequence coverage for proteins simon jaag  2021-03-23
 

I am using Skyline (64-bit) 20.2.0.343 and want to determine the sequence coverage for all my proteins.
If I hover over the protein name in the Targets pane I can see which peptides were found for each protein but is it possible to get the % coverage of the peptide and export a list of the covered peptides from Skyline?

For this workflow I used IDA from a SCIEX QTOF 5600 system which were converted to mzXML format and as search engine Comet (pep.xml) was used (both out of the Trans-Proteomic pipline software suite).

Best regards,
Simon

 
 
Nick Shulman responded:  2021-04-04
Simon,

I will try to add a column to the Document Grid which will tell you this number.

Can you send me one of your Skyline documents with some proteins and peptides in it? With new features like this, it's always good for us to be able to see some real world data where the feature is going to be used, so we can make sure that the feature looks right.

In Skyline you can use the menu item:
File > Share
to create a .zip file containing the Skyline document and supporting files including extracted chromatograms.

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

By the way, do you know what we're supposed to do if the protein sequence has repeats in it, so that a particular peptide sequence can be found in more than one location in the protein sequence? My guess would be the peptide would get counted twice so that the % coverage is telling you the number of amino acids in the protein sequence which could belong to a peptide in the document. But, I could also imagine that the community might already have chosen a different convention as to what to do in this case.

-- Nick
 
simon jaag responded:  2021-04-05
Dear Nick

thank you for your response.
You can find the Skyline documents attached.
The MS data quality is not very good because we are just starting with proteomics workflows at our research group but for that purpose it should be sufficient.

Considering your question I am not sure. In my opinion I would assume each peptide repeat is included in the calculation of the sequence coverage.

Cheers,
Simon
 
Nick Shulman responded:  2021-04-06
Thanks for sending your file. The new "Protein Sequence Coverage" column will appear in the next update of Skyline-Daily.

Here are a couple of screenshots of what the feature will look like.

Let me know if you can think of anything that should be changed.
-- Nick
 
simon jaag responded:  2021-04-06
Thank you for the fast and competent support.

It looks great.

I am looking forward for the next update.

Cheers,
Simon
 
amita puranik responded:  2021-04-15
Hello,

I am a new user of the skyline and also coming across a similar issue. I also need % sequence coverage for the analysis that I am trying to perform. I tried to follow the steps with reference to the screenshots attached in the above communication but unable to find the %sequence coverage option.

Is it exclusive for specific users? What can I do if I want to avail this facility?

I also wanted to know if there is any possibility of method automation with Skyline. I am currently using the Shimadzu QTOF system for glycoprotein characterization. I need to get multiple sets of information such as %sequence coverage, digestion efficiency, post-translational modifications, etc.

Can someone please guide me if it is possible with Skyline?

Many thanks in advance.

Regards,
Amita
 
Nick Shulman responded:  2021-04-16
Amita,
The % sequence coverage column is a new feature which first became available in the Skyline-Daily that was released this morning.

There is a button to download "Skyline-daily (beta)" on the main Skyline page:
https://skyline.ms/project/home/software/Skyline/begin.view

What exactly do you mean by "method automation"?
It has always been possible to control Skyline from the command-line and right now we are working on a new tool called "Skyline Batch" which will make writing scripts for Skyline's command-line easier.

You can learn more about Skyline batch here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=skyline-batch

-- Nick
 
amita puranik responded:  2021-04-17
Thank you so much for your fast and apt response, Sir.

By method automation, I was targeting processing multiple samples with some fixed data processing parameters. I think as you referred correctly, skyline batch could work for the same.

Many thanks again.

-Amita
 
Victor responded:  2021-07-08
I would like to expand a bit on this discussion and explore an option of adding graphic representation of protein sequence coverage as available in all major protein characterization softwares. It is nice to have a number in the column , but even better to have all those peptides visualized in regards to their spatial location within the sequence as depicted in the attached file. Cheers.
 
Brendan MacLean responded:  2021-07-08
Since it may be a while before we implement what you have presented, I will note that you can get this from Panorama, if you upload your Skyline document to a Panorama server, such as PanoramaWeb.org.
 
Victor responded:  2021-07-09
Thanks Brendan, out of sheer curiosity would it work with multiple enzymes or it is a step too far ? As for current implementation in Panorama (enclosed) just as a suggestion would it be possible in the future to set-up correlation between either peptide ID confidence or peptide XIC intensity with the colour of the gradient. I think this would immediately allow visual QC assessment and focus on weakest link so to speak. Cheers