Heavy labeled peptides (standard type in MSstats input)

Heavy labeled peptides (standard type in MSstats input) danielacgranato  2019-01-31

I have exported from Skyline the excel file in MSstats input format (SET1.csv). I would like to use it for sample size calculation in MSstats, using R. My experimental design contains heavy synthetic labeled peptides as reference peptides plus iRT spiked in to the protein digest. I would like to use this data that was obtained by SRM in order to predict the number of experimental replicates for the next experiment.

Unfortunantely, our results using MSstats in Skyline and also in R indicate a large number of samples even with a low power size (80%) and a FC of 2. I am starting to believe it is because we are not indicating properly the heavy peptides and the iRT in the Syline document. How can we indicate in the document grid that the heavy peptides are our normalizers of the endogenous counterpart and that the iRT is our control for RT drifts. Should I set iRT as global standard ? How can I set the heavy peptides as normalizers in Skyline?

Thank you very much in advace. Best, Daniela

Nick Shulman responded:  2019-01-31
I don't know the answer to this.
You should ask this question on the MSstats Google Support Group:

My understanding is that MSstats always does ratio to heavy normalization if MSstats sees that there is more than one unique value in the "IsotopeLabelType" column.
I am not sure what happens in your case, where you have both heavy peptides and Global Standards. (I think it effectively has no effect, since the global standard area ends up being in both the numerator and denominator when MSstats does its calculations).
Also, MSstats does not care about retention times, so the only reason to tell MSstats about your iRT peptides is so that MSstats will ignore them. MSstats filters out all of the rows where "StandardType" is not blank.

By the way, when you are generating a .csv file from Skyline to feed to MSstats, you should do:
File > Export > Report
and on the "Export Report" dialog, set "Language" to "Invariant". This will cause the column names to not have any spaces in them. Also, numbers get formatted to more decimal places. MSstats has been written so that it can handle CSV files with or without spaces, but the without spaces version is more like what MSstats gets when you use the MSstats external tool in Skyline.
-- Nick
danielacgranato responded:  2019-02-06
Dear Nick,
Thank you very much for your reply.
Basically, I would like to normalize the intensity of the endogenous peptides with the intensity of the heavy peptides (that were spiked in to my samples). Is it correct to set the heavy peptides as Global standards for that and apply normalization method relative to global satandards? How can I do that in Skyline or is it only possible in R?

You have informed me that in MSstats, the rows that are filled with the term ´´Global standards´´ are removed from the analysis, in which step does that occur?

Thank you for your help. Best, Daniela.
Nick Shulman responded:  2019-02-06
Hi, Daniela,

I am not sure I understand your question. Can you send me your Skyline document?
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files, including extracted chromatograms.

If that .zip file is less than 50MB, you can attach it to this support request.
Otherwise, you can upload it here:

What is the relationship between these heavy peptides, and the peptides that you want to measure? If you have spiked in heavy forms of the same peptides that you want to measure, then it would be unusual to call them global standards. You actually want each peptide to be normalized against the heavy form of itself, and the heavy forms of the other peptides would not figure into the calculation. In this case, you want to do "Ratio to heavy" normalization, and you don't want there to be any global standards at all.

Or, is this some collection of peptides that you spiked in that happen to be heavy but that do not have any real relationship to the other peptides in your document? In that case, yes, you would want to make them Global Standards.

I think I was mistaken when I said that Global Standards get removed from the dataset.
MSstats by default removes the iRT standards from the dataset, but the Global Standards remain there.
-- Nick