Peptide Mapping with Skyline

Peptide Mapping with Skyline RuMa  2021-09-20

Hi Skyline team,

I am trying to study the coverage of a biopharmaceuticals with Skyline. The goal is to digest the recombinant protein present in formulation and see if a full coverage can be obtained. The instrument used is a xevo qtof g2-xs and the data is acquired in MSE mode.

I would like to steer away from Biopharmalynx and use Skyline instead, but I find it hard to set parameters that can give me confidence in peptide identifications since I do not have a spectral library. I thought I could gain additional confidence with a predicted library using Prosit, but since the MSE mode does a ramp up of collision energy, a predicted library with a fixed CE was not helpful.

Do you have any suggestion, tutorial or paper I can refer to?



Brendan MacLean responded:  2021-09-20

Hi Ruma,
I guess I would ask if there is anything you can think of that you can do to increase your confidence in your peak identifications, e.g. matching labeled peptides - either a 15N labeled version of your biopharmaceuticals or K and R labeled synthetic peptides, or perhaps a dilution series that shows the intensity of your peaks vary as you would expect with the change in concentration. Run PLGS or some other ID tool that identifies a peptide and a time that it is believed to elute.

Since you have such a controlled experiment, I would look for orthogonal evidence that you are measuring what you think you are measuring.

Hope this is helpful. I don't have a particular tutorial that covers your case, unfortunately.


Brendan MacLean responded:  2021-09-20

Once you have done such an experiment to prove this once, you could then use Panorama to create a chromatogram library that you use in all future runs.

RuMa responded:  2021-09-21

Thanks Brendan, I have runs that I can search through Progenesis. I will build a library that way.