Spectral library essential in Skyline for the generation of optimum MRM method

Spectral library essential in Skyline for the generation of optimum MRM method shobhabr20  2019-05-03


Is spectral library essential in Skyline (Peptide Setting-> Library) for the generation of optimum MRM method for a peptide.

Please clarify.


Brendan MacLean responded:  2019-05-05

It is not necessary to have a library under Peptide Settings - Library for MRM. I recommend reviewing the Targeted Method Refinement tutorial:


In the end, the best library for MRM may be a library of chromatogram peak areas measured on your own triple-quadrupole instrument, as Skyline and Panorama allow you to create:


Hope this information is helpful in getting you started.


shobhabr20 responded:  2019-05-05

Hi Brendan,

Thanks for your reply.

I initially went through the "Skyline Targeted Method Editing" available ihttps://skyline.ms/_webdav/home/software/Skyline/%40files/tutorials/MethodEdit-3_7.pdf

In the above tutorial, they explain about "Creating a MS/MS Spectral Library"

Below content is taken from the above pdf:

Creating a MS/MS Spectral Library
Since the method you will create in this tutorial will be designed to measure yeast proteins, you will first
create a MS/MS spectral library from part of a yeast data set downloaded from the Peptide Atlas on-line
2 data repository. You can do the same for any data set from Peptide Atlas, or you can just use the full
SpectraST libraries supplied by Peptide Atlas. There are 3 large public sources of spectral libraries all
supported by Skyline:

Now I have a query , in which scenario we would use the spectral libraries..

Kindly clarify.


Brendan MacLean responded:  2019-05-06

The large spectral libraries can be useful to gain a first approximation of the relative fragment ion abundances you should expect to see in SRM/MRM, especially for y-ions. There are several papers about this, but here is one from our group:


However, since you frequently don't know the instrument on which these library spectra were measured, they will not be as accurate in the long-run as measurements on your own instrument.

Here is a paper with some insights on generating your own spectral libraries, but on a scale large enough to support DIA/SWATH, which is likely not necessary for SRM/MRM. If you have synthetic peptides, researchers will often start by measuring the synthetic peptides of interest to get high-quality MS/MS library spectra for the peptides that will be targeted.

Using public libraries is generally seen as a good starting point when you need something, but not a complete substitute for your own empirical measurements.

Some groups have taken the concept much further, though, using further experimentation to develop robust assays, fully characterized over several experiments for linear range, repeatability, etc. such as the CPTAC Assay Portal:


In summary, you should give some thought to the source of your library spectra and how closely you expect it to match your experimental conditions and how you can get out of the library measurements in prior knowledge for your own experiments.

Hope this helps. Sorry the answer leaves so much to interpretation, but that is the state of the field.