Usage

Usage modes

MS²PIP has various usage modes that each can be accessed through the command-line interface, or through the Python API.

`predict-single`

In this mode, a single peptide spectrum is predicted with MS²PIP and optionally plotted with spectrum_utils. For instance,

ms2pip predict-single "PGAQANPYSR/3" --model TMT --plot

results in:

`predict-batch`

Provide a list of peptidoforms (see Peptides / PSMs) to predict multiple spectra at once. For instance,

ms2pip predict-batch peptides.tsv --model TMT

results in a file peptides_predictions.csv with the predicted spectra.

`predict-library`

Predict spectra for a full peptide search space generated from a protein FASTA file. Various peptide search space parameters can be configured to control the peptidoforms that are generated. See ms2pip.search_space for more information.

Minimal example:

ms2pip predict-library proteins.fasta

This mode was first developed in collaboration with the ProGenTomics group for the MS²PIP for DIA project.

`correlate`

Predict spectrum intensities for a list of peptides and correlate them with observed intensities from a spectrum file. This mode is useful for evaluating MS²PIP models or for (re)scoring peptide-spectrum matches.

For instance:

ms2pip correlate results.sage.tsv --spectrum-file spectra.mgf

`get-training-data`

Given a list of peptides and corresponding spectra, generate training data for MS²PIP. This includes observed intensities for the supported ion types and the feature vectors for each ion. For more info, see Training new MS²PIP models.

`annotate-spectra`

Given a list of peptides annotate the peaks in the corresponding spectra.

Input

Peptides / PSMs

PSM file types

For peptide information input, MS²PIP accepts any file format that is supported by psm_utils. See Supported file formats for the full list. The simplest format is a tab-separated file with at least the columns peptidoform and spectrum_id present.

peptidoform is the full ProForma 2.0 notation including amino acid modifications and precursor charge state.
spectrum_id should match the TITLE or nativeID field of the related spectrum in the optional MGF or mzML file, if provided. Otherwise, any value is accepted.

For example:

peptidoform spectrum_id
RNVIM[Oxidation]DKVAK/2     1
KHLEQHPK/2  2
...

See psm_utils.io.tsv for the full specification.

Peptide sequence properties

Peptides must be strictly longer than 2 and shorter than 100 amino acids and cannot contain the following amino acid one-letter codes: B, J, O, U, X or Z. Peptides not fulfilling these requirements will be filtered out and will not be reported in the output.

Amino acid modifications

Amino acid modification labels must be resolvable to a known mass shift. This means that accepted labels are:

A name or accession from an controlled vocabulary, such as Unimod or PSI-MOD. (e.g., Oxidation, U:Oxidation, U:35, MOD:00046…)
An elemental formula (e.g, Formula:C12H20O2)
A mass shift in Da (e.g., +15.9949)

Any unresolvable modification will result in an error. If needed, PSM files can be converted with psm_utils.io and modifications can be renamed with the rename_modifications() method.

Spectrum file

In the correlate and get-training-data usage modes, an MGF or mzML file with observed spectra must be provided to MS²PIP.

Make sure that the PSM file spectrum_id matches the MGF TITLE field or mzML nativeID fields. If the values of these fields are different, but the PSM file spectrum_id is embedded in them, the spectrum_id_pattern argument can be used to extract the spectrum_id from the TITLE or nativeID fields with a regular expression pattern. For example, if an MGF entry has TITLE=scan=1, but the PSM file has spectrum_id=1, spectrum_id_pattern can be set to scan=(\d+). Note that the pattern must contain a single matching group that captures the spectrum_id.

Note

Find out more about regular expression patterns and try them on regex101.com. You can try out the above examples at https://regex101.com/r/TynuIe/1.

Spectra present in the spectrum file, but missing in the PSM file (and vice versa) will be skipped.

Output

MS²PIP supports various spectral library output formats, including TSV, MGF, MSP, Spectronaut CSV, BiblioSpec/Skyline SSL and MS2, and Encycopedia DLIB.

Note that the normalization of intensities depends on the output file format. In the TSV file output, intensities are log2-transformed. To “unlog” the intensities, use the following formula:

intensity = (2 ** log2_intensity) - 0.001