When Skyline needs to look up the spectrum for any particular peptide, Skyline goes through all of the libraries that you have listed, and the first library that contains that peptide is the one that provides the spectrum.

You can also build a library which is a combination of two existing libraries. I believe in this case, BiblioSpec will choose the spectrum which has the highest score to go into the merged library. The way that you merge two libraries in Skyline is to build a library, but, instead of adding peptide search results, you should choose an existing .blib file.

The set of peptides that you get when you choose "Import Fasta" is going to be slightly different than the set that you get when you do "Add All" from the spectral library viewer.

When you add from the spectral library viewer, peptides get matched to proteins without regard to enzyme digestion: if that peptide sequence is found within the protein sequence, then that peptide belongs to the protein. When importing a FASTA file, the set of peptides is found by applying the enzyme to the protein sequence, and then Skyline tries to figure out which of those peptides are in a library.

There are probably other differences between "Import Fasta" and "Add All Peptides" in terms of the settings on "Settings > Peptide Settings > Filter", but I am not sure exactly what those differences are.

Thanks Nic, will give this a go tomorrow.

One more question. Can you send me some info on how TIC based normalization is done with Skyline? Would like to compare data with median based normalization

You can tell Skyline to normalize to any number that you want by specifying the "Explicit Global Standard Area" in the Document Grid.

There is an explanation of how to do this in the last response on this support request:
https://skyline.ms/announcements/home/support/thread.view?rowId=35816

When merging two or more libraries using the .blib files do you use the redundant or non-redundant .blib? I assume it's the redundant since that seems to be the one where can append more spectra to?

The TIC based normalization is working within Skyline but have now hit a new road block. When I run MS Stats with "global standards" selected as the normalization option it stops with the following error message:

 Error in dataProcess(raw, normalization = inputnormalize, nameStandards = standardpepname, :
  global standard peptides or proteins, , is not in dataset. Please check whether 'nameStandards' input is correct or not.

I assume MS Stats looks for a set of peptides that are set as global standards rather than using the "Explicit Global Standard Area" values. Is there are work around for this?

Hi Nic,

Please check the last two questions on this thread.

1) When merging two or more libraries using the .blib files do you use the redundant or non-redundant .blib? I assume it's the redundant since that seems to be the one where can append more spectra to?

2) When I run MS Stats with "global standards" selected as the normalization option it stops with the following error message:

 Error in dataProcess(raw, normalization = inputnormalize, nameStandards = standardpepname, :
  global standard peptides or proteins, , is not in dataset. Please check whether 'nameStandards' input is correct or not.

I assume MS Stats looks for a set of peptides that are set as global standards rather than using the "Explicit Global Standard Area" values. Is there are work around for this?