An error of retention time

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An error of retention time shin  2019-03-12
 

Hi,

I tried to use slyline to analyze proteomic data.
Library making was succeeded, but extraction of chromatogram did not work.
Previously my setting have worked and I didn't change any saved settings.
Skyline showed an error that mentioned there is no valid retention time.
How do I show retention time in spectrum library?

Thanks,
Shin

 
 
Nick Shulman responded:  2019-03-13
I am not sure which error message you are seeing? Can you tell us what the error message says?
There's a copy button in the lower left corner of most Skyline error messages that copy the entire text of the message to the clipboard.

-- Nick
 
shin responded:  2019-03-13
Hi Nick,

The error message is here:
---------------------------
Skyline
---------------------------
The document specific spectral library does not have valid retention times.
Please check your peptide search pipeline or contact Skyline support to ensure retention times appear in your spectral libraries.
---------------------------

Shin
 
Nick Shulman responded:  2019-03-13
I am not entirely sure why that error message exists.
What type of peptide search results do you have?
If your peptide search results are supposed to have retention times, there might be something we can do to fix this if you send us your peptide search result files.

You can also cancel out of the Import Peptide Search wizard at the step that you got to, and do the rest of the steps manually. The Import Peptide Search Wizard has created a library (.blib file) for you.

1. After cancelling out of the Import Peptide Search wizard, you can add peptides to your document by doing:
File > Import > Fasta
and browse for the FASTA file you want to use
You could alternatively do "Edit > Insert > Fasta" if you want to paste in the text of a FASTA file.
Another way to get peptides into your document is "View > Spectral Libraries > Add All Button".

2. After you have the peptides in your document, you can do:
File > Import > Results
in order to direct Skyline to your raw files.
If Skyline complains about retention times at this step, then you should go to:
Setting > Transition Settings > Full Scan > Retention Time Filtering
and choose "Include all matching scans".

-- Nick
 
shin responded:  2019-03-13
Hi Nick,

Thank you for answering.
Your suggestions are concrete and helpful.

I used .dat files exported by mascot.
Other analysis have been succeeded by using mascot dat files in last month.
However it is not working now.
I would like to ask you to check my dat file.
How do I send?


Shin
 
Nick Shulman responded:  2019-03-13
You can upload large files here:
https://skyline.ms/files.url
 
shin responded:  2019-03-13
Thanks.
I uploaded one of my files. It is named "sample01_shin.dat"
 
Nick Shulman responded:  2019-03-13
Shin,

I believe you are seeing this error when you click "Next" on the second page of the Import Peptide Search wizard, after you have told Skyline where to find your mass spec data files.
Skyline is looking for three files called "1-1.wiff", "1-2.wiff" and "1-3.wiff", but I think Skyline would be willing to accept other extensions such as "1-1.mzML".

When you click the "Next" button, Skyline tries to make sure that all of the files that you have chosen have at least one retention time in the library that you just built. I do not know why Skyline is not able to find retention times for them.

Can you send me those three wiff files? In addition to the .wiff file, I also need the .wiff.scan file that is right next to it.

-- Nick
 
shin responded:  2019-03-14
Nick,

I uploaded three wiff files and wiff.scan files.
These files are integrated into zip file, named sample01_shin.
Could you try again?

Regards,
shin
 
Nick Shulman responded:  2019-03-14
Shin,

I cannot get that error to happen. Which version of Skyline are you using? You can use the menu item:
Help > About
to see the Skyline version number.

Can you send me a picture of what your screen looks like when you are seeing this error?
-- Nick
 
shin responded:  2019-03-14
Hi Nick,

I'm using skyline version 4.2.0.
This error code was found after library building.

Shin
 
Nick Shulman responded:  2019-03-14
You are seeing that error message because at least one of the WIFF files in that list on the Extract Chromatograms page does not have any retention times in the library you just created from your peptide search results.

I imagine it would be more helpful if Skyline told you which WIFF file(s) it was complaining about. I will try to fix this in a future release of Skyline.

It might be that the easiest way for you to figure out which WIFF file is a problem is by deleting some of the WIFF files from the directory that you are pointing Skyline at, and then seeing if you can make it through the Import Peptide Search wizard.

Another thing that you could do is send me what your Skyline document looks like at the point where you are getting the error.

That is, when you get to the "document specific spectral library does not have retention times" error, you can cancel out of the Import Peptide Search wizard.
Then you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including the spectral library (.blib) you just created.

I will see whether there is any file inside of the .blib file that does not have any retention times.
Skyline has a complicated way of matching up raw files to what's found in a .blib, since the .blib file often has references to, for instance, .mzML files, whereas you might want to extract chromatograms from a .wiff file.
Also, is there a space right before the dot in a some of your .dat file names like "1 .dat"? I wonder if that's going to confuse things.
-- Nick
 
shin responded:  2019-03-14
Hi Nick,

Thank you for your help.
I found the file which triggered an error. Analysis excepting this file worked as usual.
I really want to analyze this data. However I couldn't solve this problem.
Also I uploaded problem files. If you have any idea, please let me know.

Shin
 
Nick Shulman responded:  2019-03-14
Hi, Shin,

Actually, I was mistaken about what Skyline is complaining about.
Skyline gives you that error if you have any peptides whose retention time in the search results was less than or equal to zero minutes.
The peptide "HQTEGGGTQFVSLSDK" seems to have been found at zero minutes in some of those wiff files.
That error seems bogus to me, and I will try to figure out whether we can get rid of it.

After you get that error, the Import Peptide Search ends. You can then do the rest of the steps yourself.

Those steps are:
File > Import > FASTA
and point Skyline at a FASTA file containing the proteins you want to analyze.

Then, you can do:
File > Import > Results
and tell Skyline to extract chromatograms from all of the WIFF files that you have.

-- Nick
 
shin responded:  2019-03-15
Hi Nick,

I tried your method using FASTA format and succeeded to read wiff files!!
Thank you so much. I'm happy to progress research.

Regards,
Shin