MS Stats with heavy peptides

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MS Stats with heavy peptides io  2021-08-11
 

Hi all,
I am running MS Stats in a SRM experiment with heavy peptides but I think something is wrong because in the Comparison plot I see the log2 Fold Change instead of Log 2 Ratio (L/H). Maybe the label type is not correct (light instead of L, heavy instead of H) but I do not know how to change it.
I share the skyline doc
Thank you in advance
Irene

 
 
Nick Shulman responded:  2021-08-11
I don't understand. Wouldn't you expect to see "Log 2 Fold Change" in the comparison plot?

The group comparison plot will always show you a fold change of the observed abundances between the two groups. "Fold Change" means the same as "Ratio".
When MSstats sees that you have heavy peptides in your dataset, MSstats always assumes that you want to do "ratio to heavy" normalization.
My understanding is that MSstats does this sort of normalization regardless of what you might choose as the "Normalization Method" in the MSstats Group Comparison dialog. If two unique values appear in the "Isotope Label Type" column of the MSstats Input report, the normalization method is going to perform normalization by taking the ratio of the values of the two label types. I believe the code where MSstats does this is here:
https://github.com/Vitek-Lab/MSstats/blob/master/R/utils_checks.R#L144
(I cannot tell from looking at the code how MSstats decides which label type is the numerator and which is the denominator, but MSstats will give you a ratio regardless of what you name your label types.)

Therefore, the numbers that MSstats ends up calculating for each replicate really are the Light : Heavy ratios.
After MSstats has calculated the light:heavy ratios for everything, MSstats then calculates the fold changes between your two groups of replicates. So, the fold change ends up being a ratio of light:heavy ratios.

I am not sure whether there is some place to see the light:heavy values that MSstats calculates. I think those values would be called "Abundance", and it's possible that they get output to a .csv file somewhere, but I do not know where that would be.

You might get a better answer if you ask this question on the MSstats support board.
https://groups.google.com/g/msstats

-- Nick