Hi,
Without knowing what your tag is, I can't say for sure that you have the masses correct, but generally it looks like you have a correct setup for this kind of search. Unless you expect there to be lots of multiply-citrullinated peptides, I would maybe recommend removing the variable mod R 324.151 or reducing it to only 1.

To view which PSMs are found with various diagnostic/remainder ions, use the Diagnostic Feature Extraction section on the PTMs tab and enter the same masses there. It will generate a "diagnosticIons.tsv" file in the ptm-shepherd-output folder with a report of all PSMs and the intensities of all diagnostic/remainder ions you specified for each one. However, you will have to change Report mass shift as a variable mod to "No" for that method to work, so it should be done as a separate run from any quant you are trying to do.

Best,
Dan

Hi Dan,
Thanks for your help! When I set the diagnostic ion, how accurate should it be? How many digits should I have after decimal point? Can I set a ppm range for the diagnostic ion?
Another question would be if I remove the variable modification but use the diagnostic ion for peptide identification, where can I check the modified peptides in the output? I didn't see it... (I attached my current settings here)

Thanks!
Yue

Hi Yue,

When I set the diagnostic ion, how accurate should it be? How many digits should I have after decimal point? Can I set a ppm range for the diagnostic ion?

The mass tolerance for diagnostic ion detection is the same as the "Fragment mass tolerance (ppm)" setting in the PTM Profiling section of the PTMs tab. You can set this to whatever you like, but need to make sure that you provide an accurate enough mass to capture an ion within the tolerance (e.g., the ion 100.25 would not be captured with 20ppm tolerance if you provide the mass just as 100).

if I remove the variable modification but use the diagnostic ion for peptide identification, where can I check the modified peptides in the output?

If you have set "Report mass diff as a variable mod" in the MSFragger settings, the modified peptides will have modifications reported in the Assigned Modifications column. If not, modifications will be reported as mass values in the Delta Mass column. Also note that the diagnostic ions in the PTMs tab are not used for peptide identification, their results are in the diagnosticIons.tsv file in the ptm-shepherd-output folder.

Best,
Dan

Thanks Dan!
And what does Delta Mass mean?

Delta mass is the difference between observed and calculated peptide mass, which for a mass offset search is the mass of the mass offset (and the mass error and isotope error, if applicable).

Best,
Dan

Sounds good. So when we set the mass offset in the advanced options of MSFragger, it is for diagnostic ion or for the modification tag on citrullinated peptides. I am kind of confused. And if it is the mass offset for diagnostic ion, since we have two diagnostic ion, can I set two?

The mass offset is the modification itself, whereas the diagnostic ion is for determining which spectra to search for that modification. Please see our documentation or the MSFragger labile paper for further details.

There is no limit to the number of diagnostic ions you can provide.

Best,
Dan

Hi Dan,
Thanks so much for your help. Now i have some new problems.
When I want to run it, it shows "Cancelling 19 remaining tasks" and cannot go ahead.
Here is the log files, do you know how to let it work again?
log_2024-07-03_14-15-59.txt

From the log: It is a closed search while localize_delta_mass = 1. Please change localize_delta_mass to 0.

It looks like you did not specify any mass offsets in the search settings for MSFragger. Double check the parameters on the MSFragger tab if you want to do a mass offset search (which is needed if you want to use diagnostic ions). Alternatively, if you in fact want to do a closed search, load the default workflow and modify from there.

Best,
Dan

hi Dan, I just want to try new variable modification. So I chose default. But it still doesn't work. Can you help me check it?
log_2024-07-08_11-04-04.txt

Thanks,
Yue

From the log:

restrict_deltamass_to string included capital letters. Please change it to 'all' to prevent interpreting as 'resides A, L, L are allowed'.

If you follow this instruction (change "ALL" to "all"), it will resolve the issue.

Best,
Dan

Hi I change "ALL" to "all", but it stop again. I have no idea. Can you help me check again?
log_2024-07-08_16-14-59.txt

This is an issue with MSBooster - for a resolution, please submit it as a new issue since it is not related to the rest of this ticket.

However, I recommend disabling MSBooster for this search because the spectrum and retention time predictions for your modified peptides are not likely to be good (the modification is not in the training data), and it may result in reduced identification of modified peptides compared to standard scoring.

Best,
Dan

Hi Yue,

I agree with Dan that MSBooster may not help much with citrullinated peptides. If you wish to test, please download FragPipe v22, which has un updated MSBooster that resolves this error.

Best,
Kevin

Hi I disabled MSBooster, but it stops again. Could you please help me check it again?

log_2024-07-09_08-47-18.txt

Thanks,
Yue

It was due to version incompatibility. Please upgrade FragPipe to 22.0.

Best,

Fengchao

Thanks! I can successfully run it now.
I tried to use diagnostic ion to search again. But when I set "Restrict delta mass" to R, some peptides without R also show in diagnosticion.tsv file. Obviously they are not citrullinated peptides. And what do the last five column mean in this file. How could I identify citrullinated peptides.

All PSMs are shown in the diagnosticIons.tsv file for reference, but you can filter on the "Mass Shift" column to only show those with mass shifts of 324, i.e., citrullinated peptides.

Columns starting with "ox_" indicate a diagnostic ion at the given m/z value, and report the relative intensity. The Deltascore and Localization columns for a given mass are for the remainder fragment ions, indicating the increase in hyperscore when considering the remainder ion and best position(s) in the peptide based on the fragment ion(s) found, respectively.

Best,
Dan