Does Skyline can evaluate digestion efficacy of enzymes?

Does Skyline can evaluate digestion efficacy of enzymes? ref p  2021-04-15

In my assays, there's a need to monitor the trypsin digestion efficacy to improve the sequence coverage of the analyzed proteome.
In limited efforts, I didn't find useful materials for solving this. Does anyone can help me out?

Guihua Jia

Mike MacCoss responded:  2021-04-21

Hi Guihua,
Thanks for the email. So Skyline can be a major part of the methods you use to assess digestion. One of the things we have always advocated during our targeted proteomics courses is to monitor the digestion of your target protein in a time course experiment. I often show people the data in Figure 2 of this paper from the Hoofnagle lab ( You would obviously use Skyline to determine the peptide response at different time points during the digestion. You can also see how peptides are different from their own maximum -- some peptide respond worse the longer the digestion.

While this doesn't give a "digestion efficiency", I don't know how to do that unless you actually know what the response would be for 100% digestion in the sample matrix. One thing we have done in the past is use stable isotope labeled peptides in combination with a stable isotope labeled protein added to the sample matrix. We add them in a quantity so that if a peptide is released at 100% efficiency then the ratio of the peptides from the stable isotope labeled protein and the spiked stable isotope labeled peptides, the digestion would be ~1:1.

I have attached a figure of doing this for Apo-A1 with a 15N labeled protein and a C-terminal synthetic peptide. The digestion time course was done in different matrixes and with different amounts of protease inhibitors.

So I think it is pretty clear that you can design experiments to assess protein digestion and Skyline can be used to help visualize and interpret that data if the experiment is done well.

I hope this helps.