MS Annika Spectral Library exporter

Generate a spectral library for Spectronaut from MS Annika results.

Requirements

• You need to install python: https://www.python.org/downloads/
• We recommend at least 32 GB of memory for larger MS files!

Usage

• Install python 3.7+: https://www.python.org/downloads/
• Install requirements: pip install -r requirements.txt
• Export MS Annika CSMs from Proteome Discoverer to Microsoft Excel format. Filter out decoys beforehand and filter for high-confidence CSMs (see below).
• Convert any RAW files to *.mgf or *.mzML format, e.g. using ThermoRawFileParser.
• Set your desired parameters in config.py (see below).
• Run python create_spectral_library.py.
• If the script successfully finishes, the target spectral library should be generated with the extension _spectralLibrary.csv.
• Additionally decoy libraries are generated with the extensions:
  • _spectralLibraryDECOY_DD.csv: library with decoy-decoy crosslinks.
  • _spectralLibraryDECOY_DT.csv: library with decoy-target crosslinks.
  • _spectralLibraryDECOY_TD.csv: library with target-decoy crosslinks.
  • Decoys are generated by the reverse strategy as described by Zhang et al. here: https://doi.org/10.1021/acs.jproteome.7b00614.
• The full spectral library including all target and decoy annotations is created with extension _spectralLibraryFULL.csv.
  • This spectral library should be used with Spectronaut!

Usage with xiSearch + xiFDR

Starting with version 1.4.4 this script also supports input from
xiSearch with xiFDR. Simply use the validated CSMs file from
xiFDR (e.g. usually ending with extension CSM_xiFDR*.*.*.csv where * denotes the xiFDR version) as input for the CSMS_FILE parameter in the config.py file!

Exporting MS Annika results to Microsoft Excel

The script uses a Micrsoft Excel files as input, for that MS Annika results need to be exported from Proteome Discoverer. It is recommended to first filter results according to your needs, e.g. filter for high-confidence CSMs and filter out decoy CSMs as depicted below.

Results can then be exported by selecting File > Export > To Microsoft Excel… > Level 1: CSMs > Export in Proteome Discoverer.

Parameters

The following parameters need to be adjusted for your needs in the config.py file:

##### PARAMETERS #####

# name of the mgf or mzML file(s) containing the MS2 spectra
SPECTRA_FILE = ["20220215_Eclipse_LC6_PepMap50cm-cartridge_mainlib_DSSO_3CV_stepHCD_OT_001.mgf"]
# you can process multiple files like this:
# SPECTRA_FILE = ["20220215_Eclipse_LC6_PepMap50cm-cartridge_mainlib_DSSO_3CV_stepHCD_OT_001.mgf", "20220215_Eclipse_LC6_PepMap50cm-cartridge_mainlib_DSSO_3CV_stepHCD_OT_002.mgf"]
# name of the CSM file exported from Proteome Discoverer
CSMS_FILE = "20220215_Eclipse_LC6_PepMap50cm-cartridge_mainlib_DSSO_3CV_stepHCD_OT_001.xlsx"
# name of the experiment / run (any descriptive text is allowed)
RUN_NAME = "20220215_Eclipse_LC6_PepMap50cm-cartridge_mainlib_DSSO_3CV_stepHCD_OT_001-(1)"
# name of the sample organism that should be reported in the spectral library
ORGANISM = "Homo sapiens"
# name of the crosslink modification
CROSSLINKER = "DSSO"
# possible modifications and their monoisotopic masses
MODIFICATIONS = \
    {"Oxidation": [15.994915],
     "Carbamidomethyl": [57.021464],
     "DSSO": [54.01056, 85.98264, 103.99320]}
# modifications mapping for xiFDR sequences
MODIFICATIONS_XI = \
    {"Ccm": ["C", "Carbamidomethyl"],
     "Mox": ["M", "Oxidation"]}
# expected ion types (any of a, b, c, x, y, z)
ION_TYPES = ("b", "y")
# maximum expected charge of fragment ions
MAX_CHARGE = 4
# tolerance for matching peaks
MATCH_TOLERANCE = 0.02
# parameters for calculating iRT
iRT_PARAMS = {"iRT_m": 1.3066, "iRT_t": 29.502}
# regex pattern used for parsing scan number from the spectrum title
PARSER_PATTERN = "\\.\\d+\\."
# only take the best CSM per unique peptidoform and charge (True) or not (False)
GROUP_PRECURSORS = True
# raise an error if spectra do not contain FAIMS compensation voltage information
ERROR_ON_NO_FAIMS = True

In case you have more than one SPECTRA_FILE you can specify that like this:

##### PARAMETERS #####

# name of the mgf or mzML file(s) containing the MS2 spectra
SPECTRA_FILE = ["20220215_Eclipse_LC6_PepMap50cm-cartridge_mainlib_DSSO_3CV_stepHCD_OT_001.mzML",
                "20220215_Eclipse_LC6_PepMap50cm-cartridge_mainlib_DSSO_3CV_stepHCD_OT_002.mzML"]
# name of the CSM file exported from Proteome Discoverer
## <code omitted> ##

Post processing

For post processing and validation of Spectronaut result files, please read further here.

Known Issues

List of known issues

Citing

If you are using MS Annika please cite as described here.

License

• MIT

Contact

• micha.birklbauer@fh-hagenberg.at