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Error in DDA Search Library Build

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Error in DDA Search Library Build mcrawford  2024-08-14 12:19
 

I am attempting to perform label-free quantitation on two proteins in human macrophage cells. I prepared the samples using the EasyPep™ MS Sample Prep Kits from Thermofisher (Pierce), from the required amount of cells and passed over the cleanup columns.
I then injected the sample onto an Agilent 6545XT QToF running DDA MSMS over a 50 minute reversed-phase gradient on a peptide mapping column (fairly standard proteomic methods). The TIC itself looks quite dense, so there should not be a lack of data for the algorithm to mine.
I am attempting to build a spectral library for the samples, but keep encountering the following error: ERROR: No spectra were found for the new library.
I was first trying to build the library using the FASTA sequences for only my proteins of interest, but just to double check I also ran the search against the human proteome and encountered the same issue. I tried lowering score cut-offs and loosening mass tolerances, but nothing has prevented this error from showing up.
I have attached the error message, are there other pieces of information I should upload to help figure out the problem?
 
 
Nick Shulman responded:  2024-08-14 12:31
You can certainly send us all of your files and we can try to figure out what is going wrong.

You can package everything up into a .zip file and upload it here:
https://skyline.ms/files.url

It sounds like you have some peptide search results, some .raw files and a FASTA file.

It might also be helpful if you could send us 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 including extracted chromatograms and spectral libraries.

Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url
-- Nick
 
Nick Shulman responded:  2024-08-15 10:50
Thank you for uploading your files.
I see that you have a FASTA file with only two proteins in it.
I believe a FASTA file like that is much too small to effectively do a peptide search.
When you do a peptide search in Skyline, one of the steps is to search for decoy peptides and then use Percolator to figure out which of the target PSMs are good enough to pass the false discovery rate cutoff.

I am not sure whether anyone has mathematically determined what the minimum FASTA file size is to have any chance of Percolator succeeding. It might be a good idea if the Peptide Search wizard in Skyline prevented you from using a FASTA file that was clearly too small.

If you are only interested in a few proteins, you should still give Skyline a big FASTA file for peptides to search.
On the "Import FASTA" page of the Peptide Search wizard there is a checkbox "Import target proteins from a separate FASTA" which you can use to tell Skyline that you are only interested in a subset of the proteins from the big FASTA file.

It is possible that I have misunderstood your question. If that's the case, could you provide some screenshots so that I can see what you are trying to do in Skyline?
-- Nick