AlphaPeptDeep (PeptDeep)

• About
• License
• Installation
  • One-click GUI
  • Pip installer
  • Use GPU
  • Developer installer
• Usage
  • GUI
  • CLI
  • Python and jupyter notebooks
• Troubleshooting
• Citations
• How to contribute
• Changelog

About

AlphaPeptDeep (peptdeep for short) aims to easily build new deep learning models for shotgun proteomics studies. Transfer learning is also easy to apply using AlphaPeptDeep.

It contains some built-in models such as retention time (RT), collision cross section (CCS), and tandem mass spectrum (MS2) prediction for given peptides. With these models, one can easily generate a predicted library from fasta files.

For details, check out our publications.

For documentation, see readthedocs.

AlphaX repositories:

• alphabase: Infrastructure for AlphaX Ecosystem
• alphapept: DDA search engine
• alphapeptdeep: Deep learning for proteomics
• alpharaw: Raw data accessing
• alphaviz: MS data and result visualization
• alphatims: timsTOF data accessing

Subsequent projects of AlphaPeptDeep

• peptdeep_hla: the DL model that predict if a peptide is presented by indivudual HLA or not.

Other pre-trained MS2/RT/CCS models

• Dimethyl: the MS2/RT/CCS models for Dimethyl-labeled peptides.

Citations

Wen-Feng Zeng, Xie-Xuan Zhou, Sander Willems, Constantin Ammar, Maria Wahle, Isabell Bludau, Eugenia Voytik, Maximillian T. Strauss & Matthias Mann. AlphaPeptDeep: a modular deep learning framework to predict peptide properties for proteomics. Nat Commun 13, 7238 (2022). https://doi.org/10.1038/s41467-022-34904-3

License

AlphaPeptDeep was developed by the Mann Labs at the Max Planck Institute of Biochemistry and the University of Copenhagen and is freely available with an Apache License. External Python packages (available in the requirements folder) have their own licenses, which can be consulted on their respective websites.

Installation

AlphaPeptDeep can be installed and used on all major operating systems (Windows, macOS and Linux).

There are three different types of installation possible:

• One-click GUI installer: Choose this installation if you only want the GUI and/or keep things as simple as possible.
• Pip installer: Choose this installation if you want to use peptdeep as a Python package in an existing Python (recommended Python 3.8 or 3.9) environment (e.g. a Jupyter notebook). If needed, the GUI and CLI can be installed with pip as well.
• Developer installer: Choose this installation if you are familiar with CLI tools, conda and Python. This installation allows access to all available features of peptdeep and even allows to modify its source code directly. Generally, the developer version of peptdeep outperforms the precompiled versions which makes this the installation of choice for high-throughput experiments.

One-click GUI

The GUI of peptdeep is a completely stand-alone tool that requires no knowledge of Python or CLI tools. Click on one of the links below to download the latest release for:

• Windows
• macOS
• Linux

Older releases remain available on the release page, but no backwards compatibility is guaranteed.

Note that, as GitHub does not allow large release files, these installers do not have GPU support. To create GPU version installers, clone the source code and install GPU-version pytorch (#use-gpu), and then use release/one_click_xxx_gui/create_installer_xxx.sh to build installer locally. For example in Windows, run

cd release/one_click_windows_gui
. ./create_installer_windows.sh

Pip

PythonNET must be installed to access Thermo or Sciex raw data.

Legacy, should be replaced by AlphaRaw in the near future.

PythonNET in Windows

Automatically installed for Windows.

PythonNET in Linux

1. Install Mono from mono-project website Mono Linux. NOTE, the installed mono version should be at least 6.10, which requires you to add the ppa to your trusted sources!
2. Install PythonNET with pip install pythonnet.

PythonNET in MacOS

1. Install brew and pkg-config: brew install pkg-config 3. Install Mono from mono-project website Mono Mac
2. Register the Mono-Path to your system: For macOS Catalina, open the configuration of zsh via the terminal:

• Type nano ~/.zshrc to open the configuration of the terminal
• Append the mono path to your PKG_CONFIG_PATH: export PKG_CONFIG_PATH=/usr/local/lib/pkgconfig:/usr/lib/pkgconfig:/Library/Frameworks/Mono.framework/Versions/Current/lib/pkgconfig:$PKG_CONFIG_PATH.
• Save everything and execute . ~/.zshrc

3. Install PythonNET with pip install pythonnet.

peptdeep can be installed in an existing Python environment with a single bash command. This bash command can also be run directly from within a Jupyter notebook by prepending it with a !:

pip install peptdeep

Installing peptdeep like this avoids conflicts when integrating it in other tools, as this does not enforce strict versioning of dependancies. However, if new versions of dependancies are released, they are not guaranteed to be fully compatible with peptdeep. This should only occur in rare cases where dependencies are not backwards compatible.

TODO You can always force peptdeep to use dependancy versions which are known to be compatible with:

pip install "peptdeep[stable]"

NOTE: You might need to run pip install pip before installing peptdeep like this. Also note the double quotes ".

For those who are really adventurous, it is also possible to directly install any branch (e.g. @development) with any extras (e.g. #egg=peptdeep[stable,development-stable]) from GitHub with e.g.

pip install "git+https://github.com/MannLabs/alphapeptdeep.git@development#egg=peptdeep[stable,development-stable]"

Use GPU

To enable GPU, GPU version of PyTorch is required, it can be installed with:

pip install torch --extra-index-url https://download.pytorch.org/whl/cu116 --upgrade

Note that this may depend on your NVIDIA driver version. Run the command to check your NVIDIA driver:

nvidia-smi

For latest pytorch version, see pytorch.org.

Developer

peptdeep can also be installed in editable (i.e. developer) mode with a few bash commands. This allows to fully customize the software and even modify the source code to your specific needs. When an editable Python package is installed, its source code is stored in a transparent location of your choice. While optional, it is advised to first (create and) navigate to e.g. a general software folder:

mkdir ~/alphapeptdeep/project/folder
cd ~/alphapeptdeep/project/folder

The following commands assume you do not perform any additional cd commands anymore.

Next, download the peptdeep repository from GitHub either directly or with a git command. This creates a new peptdeep subfolder in your current directory.

git clone https://github.com/MannLabs/alphapeptdeep.git

For any Python package, it is highly recommended to use a separate conda virtual environment, as otherwise dependancy conflicts can occur with already existing packages.

conda create --name peptdeep python=3.9 -y
conda activate peptdeep

Finally, peptdeep and all its dependancies need to be installed. To take advantage of all features and allow development (with the -e flag), this is best done by also installing the development dependencies instead of only the core dependencies:

pip install -e ".[development]"

By default this installs loose dependancies (no explicit versioning), although it is also possible to use stable dependencies (e.g. pip install -e ".[stable,development-stable]").

By using the editable flag -e, all modifications to the peptdeep source code folder are directly reflected when running peptdeep. Note that the peptdeep folder cannot be moved and/or renamed if an editable version is installed. In case of confusion, you can always retrieve the location of any Python module with e.g. the command import module followed by module.__file__.

We used nbdev v2 for developers to build Python source code and docs smoothly from Python notebooks, so please do not edit .py files directly, edit .ipynb in nbdev_nbs folder instead. After installing nbdev, cd to alphapeptdeep project folder and run:

nbdev_install_hooks

to init gitconfig for nbdev. After editing the source code in .ipynb files, using nbdev_export to build python source code and nbdev_test to run all .ipynb files in nbdev_nbs for testing. Check nbdev docs for more information.

Usage

There are three ways to use peptdeep:

• GUI
• CLI
• Python

NOTE: The first time you use a fresh installation of peptdeep, it is often quite slow because some functions might still need compilation on your local operating system and architecture. Subsequent use should be a lot faster.

GUI

If the GUI was not installed through a one-click GUI installer, it can be launched with the following bash command:

peptdeep gui

This command will start a web server and automatically open the default browser:

There are several options in the GUI (left panel):

• Server: Start/stop the task server, check tasks in the task queue
• Settings: Configure common settings, load/save current settings
• Model: Configure DL models for prediction or transfer learning
• Transfer: Refine the models
• Library: Predict a library
• Rescore: Perform ML feature extraction and Percolator

CLI

The CLI can be run with the following command (after activating the conda environment with conda activate peptdeep or if an alias was set to the peptdeep executable):

peptdeep -h

It is possible to get help about each function and their (required) parameters by using the -h flag. AlphaPeptDeep provides several commands for different tasks:

• export-settings
• cmd-flow
• library
• transfer
• rescore
• install-models
• gui

Run a command to check usages:

peptdeep $command -h

For example:

peptdeep library -h

export-settings

peptdeep export-settings C:/path/to/settings.yaml

This command will export the default settings into the settings.yaml as a template, users can edit the yaml file to run other commands.

Here is a section of the yaml file which controls global parameters for different tasks:

model_url: "https://github.com/MannLabs/alphapeptdeep/releases/download/pre-trained-models/pretrained_models.zip"

task_type: library
task_type_choices:
  - library
  - train
  - rescore
thread_num: 8
torch_device:
  device_type: gpu
  device_type_choices:
    - gpu
    - mps
    - cpu
  device_ids: []

log_level: info
log_level_choices:
  - debug
  - info
  - warning
  - error
  - critical

common:
  modloss_importance_level: 1.0
  user_defined_modifications: {}
  # For example,
  # user_defined_modifications:
  #   "Dimethyl2@Any N-term": 
  #     composition: "H(2)2H(2)C(2)"
  #     modloss_composition: "H(0)" # can be without if no modloss
  #   "Dimethyl2@K":
  #     composition: "H(2)2H(2)C(2)"
  #   "Dimethyl6@Any N-term":
  #     composition: "2H(4)13C(2)"
  #   "Dimethyl6@K":
  #     composition: "2H(4)13C(2)"

peak_matching:
  ms2_ppm: True
  ms2_tol_value: 20.0
  ms1_ppm: True
  ms1_tol_value: 20.0

model_mgr:
  default_nce: 30.0
  default_instrument: Lumos
  mask_modloss: True
  model_type: generic
  model_choices:
  - generic
  - phos
  - hla # same as generic
  - digly
  external_ms2_model: ''
  external_rt_model: ''
  external_ccs_model: ''
  instrument_group:
    ThermoTOF: ThermoTOF
    Astral: ThermoTOF
    Lumos: Lumos
    QE: QE
    timsTOF: timsTOF
    SciexTOF: SciexTOF
    Fusion: Lumos
    Eclipse: Lumos
    Velos: Lumos # not important
    Elite: Lumos # not important
    OrbitrapTribrid: Lumos
    ThermoTribrid: Lumos
    QE+: QE
    QEHF: QE
    QEHFX: QE
    Exploris: QE
    Exploris480: QE
  predict:
    batch_size_ms2: 512
    batch_size_rt_ccs: 1024
    verbose: True
    multiprocessing: True

The model_mgr section in the yaml defines the common settings for MS2/RT/CCS prediction.

cmd-flow

peptdeep cmd-flow ...

Support CLI parameters to control global_settings for CLI users. It supports three workflows: train, library or train library, controlled by CLI parameter --task_workflow, for example, --task_workflow train library. All settings in global_settings are converted to CLI parameters using -- as the dict level indicator, for example, global_settings["library"]["var_mods"] corresponds to --library--var_mods. See test_cmd_flow.sh for example.

There are three kinds of parameter types:

1. value type (int, float, bool, str): The CLI parameter only has a single value, for instance: --model_mgr--default_instrument 30.0.
2. list type (list): The CLI parameter has a list of values seperated by a space, for instance --library--var_mods "Oxidation@M" "Acetyl@Protein_N-term".
3. dict type (dict): Only three parameters are dict type, --library--labeling_channels, --model_mgr--transfer--psm_modification_mapping, and --common--user_defined_modifications. Here are the examples: - --library--labeling_channels: labeling channels for the library. Example: --library--labeling_channels "0:Dimethyl@Any_N-term;Dimethyl@K" "4:xx@Any_N-term;xx@K" - --model_mgr--transfer--psm_modification_mapping: converting other search engines' modification names to alphabase modifications for transfer learning. Example: --model_mgr--transfer--psm_modification_mapping "Dimethyl@Any_N-term:_(Dimethyl-n-0);_(Dimethyl)" "Dimethyl@K:K(Dimethyl-K-0);K(Dimethyl)". Note that X(UniMod:id) format can directly be recognized by alphabase. - --common--user_defined_modification: user defined modifications. Example:--common--user_defined_modification "NewMod1@Any_N-term:H(2)2H(2)C(2)" "NewMod2@K:H(100)O(2)C(2)"

library

peptdeep library settings_yaml

This command will predict a spectral library for given settings_yaml file (exported by export-settings). All the essential settings are in the library section in the settings_yaml file:

library:
  infile_type: fasta
  infile_type_choices:
  - fasta
  - sequence_table
  - peptide_table # sequence with mods and mod_sites
  - precursor_table # peptide with charge state
  infiles: 
  - xxx.fasta
  fasta:
    protease: 'trypsin'
    protease_choices:
    - 'trypsin'
    - '([KR])'
    - 'trypsin_not_P'
    - '([KR](?=[^P]))'
    - 'lys-c'
    - 'K'
    - 'lys-n'
    - '\w(?=K)'
    - 'chymotrypsin'
    - 'asp-n'
    - 'glu-c'
    max_miss_cleave: 2
    add_contaminants: False
  fix_mods: 
  - Carbamidomethyl@C
  var_mods:
  - Acetyl@Protein N-term
  - Oxidation@M
  special_mods: [] # normally for Phospho or GlyGly@K
  special_mods_cannot_modify_pep_n_term: False
  special_mods_cannot_modify_pep_c_term: False
  labeling_channels: {}
  # For example,
  # labeling_channels:
  #   0: ['Dimethyl@Any N-term','Dimethyl@K']
  #   4: ['Dimethyl:2H(2)@Any N-term','Dimethyl:2H(2)@K']
  #   8: [...]
  min_var_mod_num: 0
  max_var_mod_num: 2
  min_special_mod_num: 0
  max_special_mod_num: 1
  min_precursor_charge: 2
  max_precursor_charge: 4
  min_peptide_len: 7
  max_peptide_len: 35
  min_precursor_mz: 200.0
  max_precursor_mz: 2000.0
  decoy: pseudo_reverse
  decoy_choices:
  - pseudo_reverse
  - diann
  - None
  max_frag_charge: 2
  frag_types:
  - b
  - y
  rt_to_irt: True
  generate_precursor_isotope: False
  output_folder: "{PEPTDEEP_HOME}/spec_libs"
  output_tsv:
    enabled: False
    min_fragment_mz: 200
    max_fragment_mz: 2000
    min_relative_intensity: 0.001
    keep_higest_k_peaks: 12
    translate_batch_size: 1000000
    translate_mod_to_unimod_id: False

peptdeep will load sequence data based on library:infile_type and library:infiles for library prediction. library:infiles contains the list of files with library:infile_type defined in library:infile_type_choices:

• fasta: Protein fasta files, peptdeep will digest the protein sequences into peptide sequences.
• sequence_table: Tab/comma-delimited txt/tsv/csv (text) files which contain the column sequence for peptide sequences.
• peptide_table: Tab/comma-delimited txt/tsv/csv (text) files which contain the columns sequence, mods, and mod_sites. peptdeep will not add modifications for peptides of this file type.
• precursor_table: Tab/comma-delimited txt/tsv/csv (text) files which contain the columns sequence, mods, mod_sites, and charge. peptdeep will not add modifications and charge states for peptides of this file type.

See examples:

import pandas as pd
df = pd.DataFrame({
    'sequence': ['ACDEFGHIK','LMNPQRSTVK','WYVSTR'],
    'mods': ['Carbamidomethyl@C','Acetyl@Protein N-term;Phospho@S',''],
    'mod_sites': ['2','0;7',''],
    'charge': [2,3,1],
})

sequence_table

df[['sequence']]

peptide_table

df[['sequence','mods','mod_sites']]

precursor_table

df

Columns of proteins and genes are optional for these txt/tsv/csv files.

peptdeep supports multiple files for library prediction, for example (in the yaml file):

library:
  ...
  infile_type: fasta
  infiles:
  - /path/to/fasta/human.fasta
  - /path/to/fasta/yeast.fasta
  ...

The library in HDF5 (.hdf) format will be saved into library:output_folder. If library:output_tsv:enabled is True, a TSV spectral library that can be processed by DIA-NN and Spectronaut will also be saved into library:output_folder.

transfer

peptdeep transfer settings_yaml

This command will apply transfer learning to refine RT/CCS/MS2 models based on model_mgr:transfer:psm_files and model_mgr:transfer:psm_type. All yaml settings (exported by export-settings) related to this command are:

model_mgr:
  transfer:
    model_output_folder: "{PEPTDEEP_HOME}/refined_models"
    epoch_ms2: 20
    warmup_epoch_ms2: 10
    batch_size_ms2: 512
    lr_ms2: 0.0001
    epoch_rt_ccs: 40
    warmup_epoch_rt_ccs: 10
    batch_size_rt_ccs: 1024
    lr_rt_ccs: 0.0001
    verbose: False
    grid_nce_search: False
    grid_nce_first: 15.0
    grid_nce_last: 45.0
    grid_nce_step: 3.0
    grid_instrument: ['Lumos']
    psm_type: alphapept
    psm_type_choices:
      - alphapept
      - pfind
      - maxquant
      - diann
      - speclib_tsv
    psm_files: []
    ms_file_type: alphapept_hdf
    ms_file_type_choices:
      - alphapept_hdf
      - thermo_raw
      - mgf
      - mzml
    ms_files: []
    psm_num_to_train_ms2: 100000000
    psm_num_per_mod_to_train_ms2: 50
    psm_num_to_test_ms2: 0
    psm_num_to_train_rt_ccs: 100000000
    psm_num_per_mod_to_train_rt_ccs: 50
    psm_num_to_test_rt_ccs: 0
    top_n_mods_to_train: 10
    psm_modification_mapping: {} 
    # alphabase modification to modifications of other search engines
    # For example,
    # psm_modification_mapping:
    #   Dimethyl@Any N-term: 
    #     - _(Dimethyl-n-0)
    #     - _(Dimethyl)
    #   Dimethyl:2H(2)@K: 
    #     - K(Dimethyl-K-2)
    #   ...

For DDA data, peptdeep can also extract MS2 intensities from the spectrum files from model_mgr:transfer:ms_files and model_mgr:transfer:ms_file_type for all PSMs. This will enable the transfer learning of the MS2 model.

For DIA data, only RT and CCS (if timsTOF) models will be refined.

For example of the settings yaml:

model_mgr:
  transfer:
    ...
    psm_type: pfind
    psm_files:
    - /path/to/pFind.spectra
    - /path/to/other/pFind.spectra

    ms_file_type: thermo_raw
    ms_files:
    - /path/to/raw1.raw
    - /path/to/raw2.raw
    ...

The refined models will be saved in model_mgr:transfer:model_output_folder. After transfer learning, users can apply the new models by replacing model_mgr:external_ms2_model, model_mgr:external_rt_model and model_mgr:external_ccs_model with the saved ms2.pth, rt.pth and ccs.pth in model_mgr:transfer:model_output_folder. This is useful to perform sample-specific library prediction.

rescore

This command will apply Percolator to rescore DDA PSMs in percolator:input_files:psm_files and percolator:input_files:psm_type. All yaml settings (exported by export-settings) related to this command are:

percolator:
  require_model_tuning: True
  raw_num_to_tune: 8

  require_raw_specific_tuning: True
  raw_specific_ms2_tuning: False
  psm_num_per_raw_to_tune: 200
  epoch_per_raw_to_tune: 5

  multiprocessing: True

  top_k_frags_to_calc_spc: 10
  calibrate_frag_mass_error: False
  max_perc_train_sample: 1000000
  min_perc_train_sample: 100

  percolator_backend: sklearn
  percolator_backend_choices:
    - sklearn
    - pytorch
  percolator_model: linear
  percolator_model_choices:
    pytorch_as_backend:
      - linear # not fully tested, performance may be unstable
      - mlp # not implemented yet
    sklearn_as_backend:
      - linear # logistic regression
      - random_forest
  lr_percolator_torch_model: 0.1 # learning rate, only used when percolator_backend==pytorch 
  percolator_iter_num: 5 # percolator iteration number
  cv_fold: 1
  fdr: 0.01
  fdr_level: psm
  fdr_level_choices:
    - psm
    - precursor
    - peptide
    - sequence
  use_fdr_for_each_raw: False
  frag_types: ['b_z1','b_z2','y_z1','y_z2']
  input_files:
    psm_type: alphapept
    psm_type_choices:
      - alphapept
      - pfind
    psm_files: []
    ms_file_type: alphapept_hdf
    ms_file_type_choices:
      - alphapept_hdf
      - thermo_raw # if alpharaw is installed
      - mgf
      - mzml
    ms_files: []
    other_score_column_mapping:
      alphapept: {}
      pfind: 
        raw_score: Raw_Score
      msfragger:
        hyperscore: hyperscore
        nextscore: nextscore
      maxquant: {}
  output_folder: "{PEPTDEEP_HOME}/rescore"

Transfer learning will be applied when rescoring if percolator:require_model_tuning is True.

The corresponding MS files (percolator:input_files:ms_files and percolator:input_files:ms_file_type) must be provided to extract experimental fragment intensities.

install-models

peptdeep install-models [--model-file url_or_local_model_zip] --overwrite True

Running peptdeep for the first time, it will download and install models from models on github defined in ‘model_url’ in the default yaml settings. This command will update pretrained_models.zip from --model-file url_or_local_model_zip.

It is also possible to use other models instead of the pretrained_models by providing model_mgr:external_ms2_model, model_mgr:external_rt_model and model_mgr:external_ccs_model.

Python and Jupyter notebooks

Using peptdeep from Python script or notebook provides the most flexible way to access all features in peptdeep.

We will introduce several usages of peptdeep via Python notebook:

• global_settings
• Pipeline APIs
• ModelManager
• Library Prediction
• DDA Rescoring
• HLA Peptide Prediction

global_settings

Most of the default parameters and attributes peptdeep functions and classes are controlled by peptdeep.settings.global_settings which is a dict.

from peptdeep.settings import global_settings

The default values of global_settings is defined in default_settings.yaml.

Pipeline APIs

Pipeline APIs provides the same functionalities with CLI, including library prediction, transfer learning, and rescoring.

from peptdeep.pipeline_api import (
    generate_library,
    transfer_learn, 
    rescore,
)

All these functionalities take a settings_dict as the inputs, the dict structure is the same as the settings yaml file. See the documatation of generate_library, transfer_learn, rescore in https://alphapeptdeep.readthedocs.io/en/latest/module_pipeline_api.html.

ModelManager

from peptdeep.pretrained_models import ModelManager

ModelManager class is the main entry to access MS2/RT/CCS models. It provides functionalities to train/refine the models and then use the new models to predict the data.

Check tutorial_model_manager.ipynb for details.

Library Prediction

from peptdeep.protein.fasta import PredictSpecLibFasta

PredictSpecLibFasta class provides functionalities to deal with fasta files or protein sequences and spectral libraries.

Check out tutorial_speclib_from_fasta.ipynb for details.

DDA Rescoring

from peptdeep.rescore.percolator import Percolator

Percolator class provides functionalities to rescore DDA PSMs search by pFind and AlphaPept, (and MaxQuant if output FDR=100%), …

Check out test_percolator.ipynb for details.

HLA Peptide Prediction

from peptdeep.model.model_interface import ModelInterface
import peptdeep.model.generic_property_prediction # model shop

Building new DL models for peptide property prediction is one of the key features of AlphaPeptDeep. The key functionalities are ModelInterface and the pre-designed models and model interfaces in the model shop (module peptdeep.model.generic_property_prediction).

For example, we can built a HLA classifier that distinguishes HLA peptides from non-HLA peptides, see https://github.com/MannLabs/PeptDeep-HLA for details.

Troubleshooting

In case of issues, check out the following:

• Issues. Try a few different search terms to find out if a similar problem has been encountered before.

• Discussions. Check if your problem or feature requests has been discussed before.

How to contribute

If you like this software, you can give us a star to boost our visibility! All direct contributions are also welcome. Feel free to post a new issue or clone the repository and create a pull request with a new branch. For an even more interactive participation, check out the discussions and the the Contributors License Agreement.

Changelog

See the HISTORY.md for a full overview of the changes made in each version.