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PAMPA CRAFT: Allpeptides

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touzet edited this page Apr 4, 2025 · 9 revisions

The allpeptides module allows to infer all peptides from a set of FASTA sequences through in silico digestion. Masses are automatically computed using PTM inference (see PTMs). By default, length of the peptides ranges from 10 to 30 amino-acids, the enzyme is trypsin and in silico digestion allows up to one missed cleavages. These values can be modified in the config.json file, that is included in PAMPA distribution.

As an option, allpeptide can be followed by a peak selection step when a collection of MS spectra for the same organism are available. In this case, the program will select peptides whose mass matches with peaks of the spectra.

Usage

pampa_craft --allpeptides
   -f FASTA        (multi-)FASTA file 
   -d FASTA_DIR    Directory containing (multi-)FASTA files
   -l LIMIT        Limit file that allows to select a subset of Fasta sequences 
   -s SPECTRA_DIR  Directory containing the MS spectra used to filter out peptides according to the mass 
   -e ERROR        Error margin tolerance for finding matching peaks (should be used in conjunction with -s)
   -o OUTPUT       Path of the output file (new peptide table)

Target sequences (-f, -d and -l)

The target sequences consist of amino acid sequences in FASTA format that will undergo enzymatic digestion. These sequences can be provided either as a (multi-)FASTA file (using the -f option) or as multiple FASTA files within a directory (using the -d option). The use of either -f or -d is required. Additionally, users have the option to selectively limit the set of sequences to a specific subset of organisms, molecules, or sequence identifiers using the -l option.

Option -f : The specified file can contain an arbitrary number of FASTA sequences, coming from various organisms. Refer to the [FASTA sequences](#FASTA sequences) section for details on the syntax used in FASTA headings.

Option -d: The directory can contain an arbitrary number of FASTA files, following the same requirements as with '-f' option. Only files with extension .fa or .fasta will be examined.

Option -l: This option allows to filter the set of FASTA sequences to limit the selection according to the organism (OS=), the taxid (OX=), the gene name (GN=), the sequence identifier (SeqID=). The full description of the syntax is given in section Limiting searches.

Mass spectra (-s) and error margin tolerance (-e)

Those two parameters come together and are optional. When all FASTA sequences are from a single species and mass spectra are available for that species, these parameters allow for the selection of only those peptides whose mass matches the observed peaks (up to -e). See Mass spectra for more information about the format of the spectra and the usage of -e.

Output file (-o)

This is the name of the new peptide table containing candidate peptide markers generated by the program.

Example

python3 pampa_craft.py --allpeptides -f sheep.fasta -o table_sheep.tsv

This command generates all tryptic peptides from the Ovis aries COL1A1 and COL1A2 sequences, available in sheep.fasta, and creates the corresponding peptide table table_sheep.tsv. This peptide table is ready to be used for species identification with PAMPA CLASSIFY.

Download the FASTA file: sheep.fasta

python3 pampa_craft.py --allpeptides -f sheep.fasta -s SPECTRA_SHEEP -e 0.1 -o table_sheep_filtered.tsv

This second command selects peptides that match peaks observed in at least half of the mass spectra present in SPECTRA_SHEEP. The resulting peptide table is table_sheep_filtered.tsv. Note that it allows for hydroxylation, and also for deamidations.

Download the spectra: SPECTRA_SHEEP.zip (source: https://zenodo.org/records/6967158 )

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