Error: No spectra were found for the new library -Updated request

Error: No spectra were found for the new library -Updated request Jess  2023-04-05 10:15

Edit from previous support request:
I have uploaded one DAT file to the file sharing folder named F001441.dat.

I am doing proteomic work and wanting to build a peptide spectral library using .dat files from Mascot. My software and system specifications are:

Skyline (64-bit) (28f9b9301)
Windows 10 Pro
Intel(R) Xeon(R) CPU E5-2660 v3 @ 2.60GHz

I generated mgf files from MZML files using MSConvertGUI (64-bit). My Mascot peptide searches have thousands of results. When I go into Skyline to build a spectral library, I go Settings>Peptide Settings>Library>Build. I choose my output path, and add my .dat files. Skyline then returns the error message "ERROR: no spectra were found for the new library".

I have attached 3 screenshots for 3 different instances where I received the error messages, described below:

  1. First error message after uploading my .dat files to build a peptide spectral library.

  2. After looking at different support threads on here, I tried converting my MZML files to mgfs again, this time making sure TPP compatibility was selected. I did a test search on Mascot with 1 mgf file, with over 1000 peptides being identified. To get my .dat file, I clicked "save link as" and changed the file name to end in .dat. This error message came after I uploaded that singular dat file.

  3. I thought there was an error in my dat file based on how I was exporting. Using the same TPP compatible mgf from above, I went into the search result report on Mascot and created a dat file by specifying "Export format: Mascot DAT file" and clicking Export Search Results. I did not change any file names and still got a Skyline error.

Can anyone tell me how to fix this issue? Is it a problem with my DAT file, or a problem with how I am generating my spectral library in Skyline?

Brian Pratt responded:  2023-04-06 10:34

Just a guess, but it seems likely that the trouble comes from scan information being muddied by the multiple translations raw->mzML->mgf. If you have the raw file, try skipping the mzML conversion - it's not necessary for Skyline.

Best regards,

Brian Pratt

Matt Chambers responded:  2023-04-14 06:39

It seems BiblioSpec is silently ignoring some errors from Mascot Reader. When I fixed that, I got:

Error with 'F001441.dat'. Invalid number of queries. (Invalid number of queries in results file (108586) - truncated to (49743))

Which suggests a corrupt DAT file to me. But when I look at the DAT file, it doesn't appear truncated. So I'm not sure what's going on. Can you send another example DAT? Perhaps a smaller one?

Jess responded:  2023-04-14 08:31

Hi Matt,

Unfortunately, that is the smallest DAT size we can obtain. We are doing Mascot searches to identify peptides from 24 mgf files. From those searches, we were getting 4 gb DAT files. The DAT file uploaded was a subsequent search done with a single mgf file rather than all 24, as we thought the size was the issue as well.
Although we could not get the DAT files to import, we managed to find a workaround for Skyline quantitation by directly accessing the NIST database we searched in.

Thank you for looking into this, I think we are cutting our losses for now.