Configuration

READ requires a configuration file in .toml format^[1] as input. An example READ config file is given in the config/ directory ➡️ see here. Please set the following parameters according to your needs in the config.toml file:

[METHOD]
# window size
window_size = 0.5
# window start (m/z)
window_start = 400.0
# window end (m/z)
window_end = 800.0
# window overlap
window_overlap = 0.0

[MATCHING]
# m/z tolerance for matching peaks in Dalton
mz_tolerance = 0.02
# retention time tolerance in seconds for matching identifications to MS2 spectra
rt_tolerance = 3.0
# retention time window in seconds that a MS1 and corresponding MS2 spectrum must be in
ms1_rt_window = 10.0

[ISOTOPES]
# whether precursor isotopes should be considered for purity calculation
consider_precursor_isotopes = true
# isotope match tolerance in Dalton
isotope_tolerance = 0.01
# maximum considered precursor charge
max_charge = 6

[FILTERING]
# precursor intensity fraction in the window to use as reference, only used for displaying some preliminary statistics
# for filtering use PROTEIN.min_purity
total_intensity_threshold = 0.7
# minimum relative intensity threshold compared to most intense peak in window to not be considered noise
noise_threshold = 0.1

[QUANTIFICATION]
# subtract the reporter noise from the reporter signal?
# if true, filtering by S/N should be turned off or thresholds set to 0.0
subtract_noise = true
# quantification method to use
# 1 = native
# 2 = OpenMS
# 3 = Resolution GUI
quantification_method = 2

[PROTEIN]
# Qvalue that should be used for filtering, only used for DIA-NN and Spectronaut
q_value = 0.01
# Normalized Chimerys Coefficient Threshold, anything below will be ignored, only applies to Chimerys
min_chimerys_coefficient = 1.0
# minimum average reporter S/N for a PSM to be considered for aggregation, only applies to Chimerys
min_avg_reporter_sn = 10.0
# minimum reporter resolution to be considered for aggregation
min_reporter_res = 45000.0
# minimum purity for a PSM to be considered for aggregation
min_purity = 0.7
# whether or not ambiguous protein groups should be filtered out, only used for DIA-NN and Spectronaut
keep_ambiguous_protein_groups = false

[CONDITIONS]
# please define your conditions here
# conditions should be given as condition name (without spaces) following an equal sign and then a list of TMT reporters
# see examples below
all = ["TMTpro-126",  "TMTpro-127N", "TMTpro-127C", "TMTpro-128N", "TMTpro-128C",
       "TMTpro-129N", "TMTpro-129C", "TMTpro-130N", "TMTpro-130C", "TMTpro-131N",
       "TMTpro-131C", "TMTpro-132N", "TMTpro-132C", "TMTpro-133N", "TMTpro-133C",
       "TMTpro-134N", "TMTpro-134C", "TMTpro-135N"]
cond1 = ["TMTpro-126",  "TMTpro-127N", "TMTpro-127C", "TMTpro-128N", "TMTpro-128C",
         "TMTpro-129N", "TMTpro-129C", "TMTpro-130N", "TMTpro-130C"]
cond2 = ["TMTpro-131N", "TMTpro-131C", "TMTpro-132N", "TMTpro-132C", "TMTpro-133N",
         "TMTpro-133C", "TMTpro-134N", "TMTpro-134C", "TMTpro-135N"]
# please define the min S/N thresholds per condition that should be used for protein aggregation here
# this should be sn_thresholds = map of thresholds for each condition
# see example below
sn_thresholds = { all = 0.0, cond1 = 10.0, cond2 = 10.0 }
# please define the min abundance thresholds per condition that should be used for protein aggregation here
# this should be s_thresholds = map of thresholds for each condition
# see example below (in this case -> no abundance thresholds)
# please not that the conditions have to be the same in sn_thresholds and s_thresholds
s_thresholds = { all = 0.0, cond1 = 0.0, cond2 = 0.0 }

IMPORTANT

You might also want to adapt the isotope correction factors for your TMT lot, you can do that in the tmt18plex_default.ini file. Please refer to the documentation site of OpenMS here.

Method Parameters

The following parameters can be adjusted in the [METHOD] section:

• window_size:
  • Size of the isolation window in m/z (float).
• window_start:
  • Start of the mass (m/z) range for MS scans (float).
• window_end:
  • End of the mass (m/z) range for MS scans (float).
• window_overlap:
  • Size of the isolation window overlap in m/z (float).

IMPORTANT

Please note that if a window file is provided to READ via -w or --window it will automatically override these parameters!

Matching Parameters

The following parameters can be adjusted in the [MATCHING] section:

• mz_tolerance:
  • The m/z tolerance used for matching peaks in Dalton (float).
• rt_tolerance:
  • The retention time tolerance used for matching identifications to MS2 spectra in seconds (float).
• ms1_rt_window
  • The retention time window that a MS1 and corresponding MS2 spectrum must be in, in seconds (float).

IMPORTANT

These parameters majorly control how identifications are matched to (precursor) peaks and MS spectra, usually the values in the default config file are a good selection!

Isotope Parameters

The following parameters can be adjusted in the [ISOTOPES] section:

• consider_precursor_isotopes:
  • Whether precursor isotopes should be considered for precursor co-isolation purity calculation (bool).
• isotope_tolerance:
  • Tolerance used for identifying isotope peaks in Dalton (float).
• max_charge:
  • The maximum considered precursor charge when identifying isotope peaks (int).

Filtering Parameters

The following parameters can be adjusted in the [FILTERING] section:

• total_intensity_threshold:
  • Parameter only used for logging some preliminary statistics, READ will display how many precursors pass this co-isolation purity value (float).
  • The actual filtering based on co-isolation purity is controlled via PROTEIN.min_purity!
• noise_threshold:
  • The minimum relative intensity threshold compared to the most intense peak in the isolation window to not be considered noise, e.g. 0.1 denotes that any peak below 10% intensity of the most intense peak in the isolation window is considered noise (float between [0, 1)).

Quantification Parameters

The following parameters can be adjusted in the [QUANTIFICATION] section:

• subtract_noise:
  • Whether the reporter noise should be subtracted from the reporter signal (bool).
  • If this is set to true you should (probably) not apply any signal-to-noise filters!
  • Requires the output of the TMT Resolution GUI Tool as input for READ!
• quantification_method:
  • Which quantification method to use (int between [1, 3]).
  • Can be 1 for native quantification, or 2 (recommended) for quantification with OpenMS (requires installation of OpenMS), or 3 for quantification based on the TMT Resolution GUI Tool (requires the output of the TMT Resolution GUI Tool as input for READ).

Protein Filtering and Aggregation Parameters

The following parameters can be adjusted in the [PROTEIN] section:

• q_value:
  • The Qvalue that should be used for filtering (float).
  • Only used for DIA-NN and Spectronaut, Chimerys reports are assumed to be filtered/validated.
• min_chimerys_coefficient:
  • The minimum normalized Chimerys Coefficient, anything below will be ignored (float).
  • Only used for Chimerys.
• min_avg_reporter_sn:
  • The minimum average reporter S/N for a PSM to be considered for aggregation (float).
  • Only used for Chimerys.
• min_reporter_res:
  • The minimum reporter resolution for a reporter to be considered for aggregation (float).
  • Requires the output of the TMT Resolution GUI Tool as input for READ to be applied! Otherwise parameter is ignored!
• min_purity:
  • The minimum co-isolation purity for a PSM/precursor to be considered for aggregation (float between [0, 1]).
• keep_ambiguous_protein_groups:
  • Whether or not ambiguous protein groups should be filtered out (bool).
  • Only used for DIA-NN and Spectronaut.

Condition Parameters

The [CONDITIONS] section allows setup of different conditions based on the reporter ions. Each condition should be given as the condition name (without spaces) followed by an equal sign and then a list of TMT reporter ions, for example:

all = ["TMTpro-126",  "TMTpro-127N", "TMTpro-127C", "TMTpro-128N", "TMTpro-128C",
       "TMTpro-129N", "TMTpro-129C", "TMTpro-130N", "TMTpro-130C", "TMTpro-131N",
       "TMTpro-131C", "TMTpro-132N", "TMTpro-132C", "TMTpro-133N", "TMTpro-133C",
       "TMTpro-134N", "TMTpro-134C", "TMTpro-135N"]

This would define one condition called all which contains all reporter ions.

The following parameters can then be adjusted in the [CONDITIONS] section:

• sn_thresholds:
  • The minimum S/N thresholds per condition that should be applied for protein aggregation.
  • For example: sn_thresholds = { all = 0.0 }
    (no minimum S/N filter for condition all).
• s_thresholds:
  • The minimum abundance thresholds per condition that should be applied for protein aggregation.
  • For example: s_thresholds = { all = 0.0 }
    (no minimum abundance filter for condition all).

TIP

Please also check the example config file for a better demonstration on how to set up different conditions!

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1. https://toml.io/ ↩︎