Missing 'Assigned Modifications' for N-terminal mods in detailed mass-offset search

[tjlundgren]

Hello Fragpipe team,

I am trying to analyze results from a mass-offset search. In the psm.tsv output, there are some PSMs with a reported 'Modified Peptide' but no 'Assigned Modification'. These only occur when the modified aa is at the N-terminus of the peptide, but not all N-terminal modifications are missing the 'Assigned Modification'.

Is there a reason some modified peptides are not given an 'Assigned Modification' output?

Note the option to 'report mass shift as a variable mod' is set 'Yes, remove delta mass' to make IonQuant happy, but I am trying to calculate the precise total delta mass (including shift) for filtering the data. I have done this with the 'Assigned Modification' output, but the integer value in 'Modified Peptide' alone may not be sufficient.

Fragger params and example psm.tsv: https://drive.google.com/file/d/1cGJI8GxfUTs30An2zvGZdfJPxZroWtD8/view?usp=drive_link

[dpolasky]

Hi, MSFragger 4.2 does not support mass offsets at peptide/protein termini. We have a prerelease version that does support offsets at termini and fixes this issue - you can email me directly at dpolasky [at] umich [.] edu if you want to try it out.

Best, Dan

[tjlundgren]

Thanks for the quick response Dan,

To clarify - the example psm.tsv is the result of a search for mass-offsets without any termini-specific designations. Some of these location-generic modifications were localized/assigned to the peptide N-terminus, presumably causing the 'Modified Peptide' PSMs with no 'Assigned Modifications'. By 'does not support mass offsets at peptide/protein termini', do you mean support for modification definitions restricted to the termini?

I am still unclear about my original question - Is there a reason some modified peptides are not given an 'Assigned Modification' output in mass-offset search? Do I need to disregard/remove all mass-offsets assigned to a peptide terminus?

Thanks again,

fraggerparams.txt

[dpolasky]

I see - yes, the mismatch in modified peptide/assign mods is a bug. I believe it was fixed in the prerelease version as a result of the changes we made to support terminal offsets, but let me know if that's not the case.

Best, Dan

[tjlundgren]

Alas, MSFragger v4.2_rc-4 also has many 'Modified Peptide' without an 'Assigned Modification', also only occurring at the peptide N-terminus. psm_truncated.zip Edit: Here is a truncated psm from my mass-offset search, with only PSMs that have a 'Modified Peptide' entry

Also, this added some confusing annotations in 'Modified Peptides': There are now deletion modifications? (eg. 'A[0]PEPTIDER'), and others with imaginary? modifications (eg. G[-1]ADEELVK with an adjusted delta mass of ~1; G[-19]AAFLHLTD) These examples also only occur at the N-terminus...

I don't know if it is related, let me know if I should ask elsewhere - how does Fragger resolve isotope error with mass offsets (such as deamidation, +0.984)? fraggerparams.txt