Questions about zeros in the output of experiments with multiple groups

[GuotingUH]

I'm using the FragPipe to analyze MS data and have a few questions about the many 0's in the combined peptide output file.

My experiment normally has control and treatment groups. If one peptide was identified in control group, but not in the treatment group, in the combined output file, would the intensity values for this peptide be 0 in the treatment group?

If one peptide was identified in both groups, is it possible that the peptide passed the fdr threshold in one group, but didn't in the other group? If so, would the intensity for this peptide be 0 in the group in which it didn't pass the fdr?

If the intensity values are both zeros in the above two cases, I wonder if there is some setting in the software that I can set to differentiate these two situations.

If the intensity value in one of the cases is not zero, what would the value be in that case?

Thank you very much!

[prvst]

First, let's assume that you have a label-free experiment where controls and treatments are separate. Each MS run will go through an individual peptide validation process, and a combined protein inference is then calculated (a.k.a global inference). PSMs, peptides, and ions are filtered separately, meaning that for each MS run you will have a report containing FDR-approved observations. When Philosopher assembled the combined reports, the program will only use the FDR-approved observations from each MS run, or report. If a given peptide is present in control, and not the treatment, you should see an intensity value for the control, and vice versa. Now, have in mind that a peptide might be present, but its quantification might have failed or returned 0, there are situations that can explain this.

It is also possible for a peptide to pass the FDR filtering in one MS run, and not the other. In this case, you'll see an intensity of 0 for the missing peptide.

If the peptide passed the FDR filtering, but it's quantification is missing or it's zero for both cases, then you can try adjusting the quantification parameters, increasing the quantification window.

[GuotingUH]

Thank you very much for the quick response!

If one peptide was detected and quantified (i.e. has a non-zero intensity value) but didn't pass the FDR filtering, is there a way to keep the intensity value for this peptide in the report? or where to find such intensity values for this kind of peptides?

Thanks again.

[prvst]

Try increasing the FDR threshold. The default value is 1%, try working with 3%, for example.

[GuotingUH]

Thanks