Create Background Proteome

support
Create Background Proteome Richard Lam  2019-08-13
 

Hello,

I followed the tutorial document 'MethodEdit-3_7' (see attachment, step on pg 6) to create the 'Yeast' background proteome in the 'MethodEdit' skyline document (see attachment). After I clicked 'OK" to digest the background proteome, Skyline stalled and could not proceed with the digestion (see the screen shot in the attachment (Building Background Proteome in Skyline).
I tried several time and restarted Skyline and my computer, the issue did not go away. Please help.

Regards,

Richard

 
 
Nick Shulman responded:  2019-08-14
Richard,

What exactly is happening in that screenshot that you provided? Are you saying that it is not possible to click anywhere on Skyline?

Sometimes when it is not possible to click on Skyline that is because Skyline tried to bring up a message box, but because of a bug the message box ended up appearing behind the Skyline window. If that's happening, you might be able to find the message box if you press the "Windows" and "D" keys on your keyboard, which is the keyboard shortcut to minimize all of the windows and show the desktop.

If it is possible to click on and interact with Skyline, then you should just continue with the rest of the steps of the tutorial. You do not need to wait for Skyline to finish with that "Accessing web services to retrieve details for X proteins" work.

-- Nick
 
Brendan MacLean responded:  2019-08-14
Hi Richard,
I think we need to change the description in the tutorial which currently reads:

"Skyline begins digesting the background proteome with the active protease enzyme, in this case Trypsin. Progress is reported in the status bar, and you can continue work. While this digestion is occurring, we can begin adding proteins to the document to better understand how the new spectral library impacts the peptide and transition choices Skyline makes."

What you are seeing in the status bar is not actually tracking digestion of the protein sequences with the active protease. Nick changed the flow to make all necessary "digestion" occur in the Edit Background Proteome form. So that all happened during the import of the FASTA file when you saw the progress for "Saving..."

What you now see in the progress bar is tracking Skyline using the Internet to download extra information about the yeast peptides. This can take a while but usually completes eventually. Though, as Nick points out, it should not block you doing anything else in Skyline and you should be able to just continue working. If it truly never completes, then there may be something wrong with Skyline's access to Uniprot from your computer.

Thanks for posting the screenshot. Sorry for the confusion and not keeping our tutorials completely up to date with changes in the software.

--Brendan
 
Richard Lam responded:  2019-08-14
Hi Brendan and Nick,

Actually, it never completes and remains as what's indicated in the screen shot.
I tried it in my workplace and at home and both would not complete.
Can you try it using my Skyline document and see if it works for you?

Best,

Richard
 
Brendan MacLean responded:  2019-08-14
I think you would need to post a .sky.zip (produced with File > Share - Complete) for us to be able to test this on our side. The .sky file alone does not contain the .protdb which is causing the UniProt requests that seem to be hanging for you. What version of Skyline (Help > About) are you using?
 
Richard Lam responded:  2019-08-14
Hi Brendan,

Attached, please find the .sky.zip file.
FYI, I am using Skyline (64-bit) 19.1.0.193

Thanks!

Richard
 
Brendan MacLean responded:  2019-08-14
So, for me, the progress indicator goes away after about a minute. Then if I close the file and reopen it the progress indicator comes back but reports a lower number of proteins it is seeking information for.

I am down to 4948 out of 5801.

It does seem to be making very slow progress of maybe 100 per minute or so? This seems much slower than normal, but there may be nothing we can do about that because we are querying UniProt for this information. I am now going to give it an hour or two to see if it finishes unattended or whether it actually requires me to close and reopen the document to continue making progress after the progress indicator disappears.

We will look into it and maybe contact the UniProt people to see if they can explain why their server is responding so slowly.

Thanks for the report and the file.

--Brendan
 
Richard Lam responded:  2019-08-14
Hi Brendan,

You said when I press 'OK', it will track Skyline using the Internet to download extra information about the yeast peptides (querying UniProt). What extra information it is getting from UniProt? I thought the 'sgd-yeast' FASTA file has all the protein/sequence information about the background proteome already.

Best,

Richard
 
Brendan MacLean responded:  2019-08-17
Accession Number, Gene Name, Preferred Name, Organism

Yep. If you don't need these fields, you should be good to start working and ignore this status which should go away eventually. We will look into what is going on with UniProt and this SGD FASTA file. Thought, it is August and the developer who owns this code just left on a final summer vacation.

Thanks for reporting this issue. There is definitely something wrong with this interaction, which has worked much better in the past.

--Brendan