Combined spectral library

Combined spectral library n prust  2023-04-12


I have a question regarding a combined spectral library.

When generating a spectral library containing multiple result files, is there an order in which the final spectral library entry to be matched to is picked?
Meaning if I have identified the same peptide in multiple result files that are used to generate the spectral library, how is Skyline deciding which of the spectra to use to match the PRM targets to?

I attached an example of a peptide that is identified in two result files that were used to generate one spectral library. The first result file is from a MQ search containing multiple fractions and the second result file is from a PD file. As you can see the peptide is identified multiple times and the spectra quality differs. Skyline decided to use the first entry in the spectral library for the result matching. This decision does not seem to be based on intensity, spectral quality or on alphabetical order, nor on the oder of loading the result files to generate the spectral library.

Could you provide me with some information on this decision process or if it's a random decision?


Nick Shulman responded:  2023-04-12
When you create a spectral library in Skyline, there is a two-step process.
First, a program called BlibBuild.exe creates a file whose name ends in ".redundant.blib" which contains all of the spectra.
Then, a program called BlibFilter.exe creates a file whose name ends in ".blib" which contains the best scoring spectrum for each peptide+charge from the .redundant.blib file.
BlibFilter.exe is supposed to choose the spectrum with the best score.
In the screenshots that you provided it looks like each of the spectra from the redundant library got exactly the same score (0.000004). If multiple spectra tied for the best score, then BlibFilter will choose the spectrum with the highest total intensity.
In your screenshot, it does appear that the first spectrum that you show has the highest total intensity.

I see in your screenshot that right next to most of the peaks that Skyline is drawing in blue, there is another much larger peak with a very similar m/z which Skyline is drawing in black. Those black peaks still contribute to the total intensity that BlibFilter used to choose the best spectrum, but they do not contribute to Skyline's idea of how to calculate the library dot product.
-- Nick
n prust responded:  2023-04-14
Hi Nick,

thanks for the swift reply and short explanation. :-)