Question: peptidoform vs peptide

[vvsbiocode]

Hi,

I’m a bit confused about the distinction between a peptidoform and a peptide.
My understanding is:

• Peptidoform = sequence + modifications + charge
• Peptide = sequence only

However, this seems to conflict with how MS2Rescore computes peptide-level scores (q-value, PEP). It appears to treat a peptide as sequence + modifications, stripping only the charge [ref].

Could you please advise, if I want to infer peptide-level results from MS2Rescore, should I:

• use the provided meta:peptide_score, meta:peptide_qvalue, and meta:peptide_pep columns (i.e., peptide = sequence + modifications)
• or recompute scores myself by grouping PSMs by sequence only (stripping both modifications and charge)?

Thanks!

[RalfG]

Hi @vvsbiocode,

In most cases, a peptidoform would be considered as peptide + modifications, while peptide + modifications + charge, would be seen as precursor. However, in the development of the ProForma 2 peptidoform (and proteoform) notation, the charge was optionally included to enable spectrum annotations (sometimes the annotated charge is different from the mass spec-assigned charge of the spectrum).

That is why throughout most of MS²Rescore's codebase, peptidoform instances include the precursor charge. However, for proper FDR calculation at the peptidoform level, we indeed strip the charge. Peptides are indeed always considered sequence only.

With ms2rescore<4.0.0, you can indeed directly use the provided meta:peptide_score, meta:peptide_qvalue, and meta:peptide_pep columns. Unless there was a bug, I believe Mokapot strips the modifications itself to calculate peptide-level FDR.

However, starting with v4.0.0, we have simplified the score output files at various levels:

File	Description
<prefix>.psms.tsv	PSMs and their rescored score, at PSM-level FDR.
<prefix>.peptidoforms.tsv	Peptidoforms (sequence and modifications, no charge) and their score, at peptidoform-level FDR.
<prefix>.peptides.tsv	Peptides (stripped sequence) and their score, at peptide-level FDR.
<prefix>.proteins.tsv	Protein groups and their score, at protein group-level FDR.

Best,
Ralf