PRM-quantitate with precursor or fragment?

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PRM-quantitate with precursor or fragment? sallym8  2021-01-10
 

Hi Skyline Team!

Your software and resources are amazing- thank you for all your hard work! I recently ran my first PRM experiment on an Orbitrap Fusion and have been analyzing my data with Skyline. In the past I have run DDA experiments, but my protein is in a buffer with albumin and my peptides of interest have been getting drowned out. I have been able to use a large amount of protein and the DDA method in order to generate a list of peptides that I could include in my PRM experiment, but moving forward I will be limited in the amount of protein I can use. The goal of my experiment is to quantitate peptides in two regions of the protein in order to monitor a cleavage event. Currently, I am trying to compare the PRM and DDA experiments in order to determine how much the new method improved my signal. I am confused about whether I quantitate the PRM data using the precursor ions or the fragment ions and why. If you could offer some clarification that would be amazing!

 
 
Nick Shulman responded:  2021-01-10
If your samples are at all complex (i.e. they contain cells), then you will probably get better numbers if you use the MS2 data for quantification. The reason for this is that in any reasonably complex mixture, there will always be other molecules which have approximately the same precursor m/z and retention time. If you quantify using the MS1 numbers, your results will be inaccurate because they include signal from molecules that you are not trying to measure. The MS2 signal is a lot smaller, but is much less likely to have interference from other molecules.

If your samples are not complex (e.g. you spiked your protein of interest into saline) then you will get better numbers if you use the MS1 signal, because the MS1 signal is so much larger than the MS2 signal and you do not need the selectivity that comes from filtering on both the precursor and product m/z's.

If you are trying to compare PRM quantification to DDA, I would expect that you would only find that PRM MS2 quantification does better if your samples are complicated enough that you really are getting interference in the MS1 signal. There is a good illustration of this happening on page 26 of the PRM tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_prm

-- Nick