ERROR: Failed to find required column named 'file'.

ERROR: Failed to find required column named 'file'. zzhu248  2022-05-26

Dear Developers and all researchers,

When I was trying to build the library via peptide setting, I upload my .ssl file and it pops up warning as title.

For the specific workflow, I am following a paper from nature protocol:
I was working on the crosslinking setting and stuck on the step 39.

I would be very appreciated for your help!

Best Regards,

Nick Shulman responded:  2022-05-26
This is the web page that says what the .ssl file is supposed to look like:
and here is an example file:

If you have trouble figuring out what you need to fix with your .ssl file, you can send us that .ssl file and we can take a look.
Files which are less than 50MB can be attached to this support request. You can upload larger files here:

If you send us the .ssl file, it would also be helpful if you could send us the raw file(s) that the ssl file is referencing.

By the way, about 2 years ago new features were added to Skyline to natively support crosslinked peptides.
You can find information about the crosslinked peptide feature in Skyline here:

In the paper that you link to, it looks like they told Skyline that their crosslinked peptide was just one big long peptide sequence. I haven't read the whole paper, but it looks like they were only quantifying using MS1 so all they needed was that the intact mass of the thing in Skyline match the whole crosslinked peptide.
-- Nick
zzhu248 responded:  2022-05-26
Hi Nick,
Thank you for your fast response. I have asked one of my lab members and he says it seems like the column "referring" to the path of every self-generated peptide is missing. I am scrutinizing my previous work.

FYI, I have attached the ssl files below. I have tried the one with only last column and it does not work as well
Nick Shulman responded:  2022-05-26
Where did you get that .ssl file? Was that a file that you downloaded from supplemental data associated with the paper, or was that a file that you created yourself?
A .ssl file needs to have columns named "File" and "Scan". When BiblioSpec is building a spectral library from a .ssl file, Skyline will look in the raw file referred to in the File column and will find the particular spectrum. That spectrum will be put into the .blib file that is being created.

Is there a reason that you are trying to recreate the steps from this paper? In the last few years, a lot of really good crosslinked peptide search engines have been developed so you should not need to resort to writing a .ssl file with a list of peptides and scan id's.

BiblioSpec can create a spectral library from crosslinked peptide search results so long as those search results have been converted to the "proxl" XML format. On the proxl web page, you can see a list of the crosslinked peptide search engines which can be converted to proxl and can therefore be used by Skyline and BiblioSpec:

-- Nick
zzhu248 responded:  2022-05-26
This is a self-created file. I was applying xiView platform from Rappsilber Lab since they did a very good job on data visualization. I am currently looking forward to a proper way to proceed quantification based on crosslinkers BS3d0 and d4. I have also tried plink and XlinkX and neither of them can provide .ssl file, unfortunately. (TBH, It tooks me 2 days to manually create that file) If you have any suggestions about this field I would be very grateful to your help!
Nick Shulman responded:  2022-05-26
I don't think you should be trying to create .ssl files.

It is possible to convert plink crosslinked peptide search results to proxl, so I think you should do that.

Here is the project page for the tool which converts plink to proxl:

In the "How to run" section it tells you where to download the file "plink2toProxlXML.jar" and how to use Java to run the jar with commandline arguments to convert a .plink file to proxl.xml

Once you have proxl.xml files you can use those to build a BiblioSpec spectral library.
In Skyline, you can use the "File > Import > Peptide Search" menu item to go through the process of creating a spectral library from proxl search results and then importing proteins from a FASTA file and extracting chromatograms.

Let us know if you get stuck trying to convert your plink search results to proxl.xml.
-- Nick
zzhu248 responded:  2022-05-26
Hi Nick,

I am afraid this does not work for me as it only accepts single linker without performing quantification. I could send them the files directly, but I would still give it a try on my previous method. Anyway, I am very much appreciated for your help and hope you have a wonderful rest of the day.