ERROR: No spectra were found for the new library

ERROR: No spectra were found for the new library Lily  2023-05-18

Hi all,
I want to build a peptide spectral library by using .raw file,and then when I use the DDA Search for MS1 Filtering of the Skyline, the results show that the search failed,so I convert the format of my fasta files to .mz5,because I find the file format of tutorials is .mz5,then the results show “No spectra were found for the new library”.
Can anyone tell me how to fix this issue?

Nick Shulman responded:  2023-05-18
I do not know what might be going wrong but if you send us your files we could try to figure it out.
You could package all of your files up in a .zip file and upload the .zip file here:

-- Nick
Lily responded:  2023-05-25
I have uploaded the relevant documents, please check.
Nick Shulman responded:  2023-05-25
Thank you for sending those files.
When I told Skyline to do a DDA search on both of the raw files that you uploaded, I got the error "Search Failed" when searching the first file.

When I told Skyline to only search the file "TEclipse_DDA_DFGDMG006_DFGDMG006_F2.raw", I was able to successfully get through the whole search process, but I did get the "Error: No spectra were found for the new library".

I think the problem is that the peptide search engine really did not find any peptides with a high enough confidence level to be included in the library.

After you have finished the peptide search, there will be some .mzid files which have the raw search results in them. The only peptide spectrum matches in the .mzid files are very low quality matches where only the y1 and y2 ions were matched.

What was in your samples? Did you spike any peptides into the samples that would be very easy for a peptide search engine to find? Are those peptides in the FASTA file?

I think the problem is that there really are no peptides that can be found in that sample, so you should try to figure out whether the problem happened during sample preparation or while the data was being acquired inside the mass spectrometer.
-- Nick