READ

[R]eporter Ion [E]xtractor and [A]nnotation [D]irector

READ: A tool and python package to orchestrate TMTpro-18plex quantification for [single cell] DIA and DDA searches with Chimerys, Spectronaut, and DIA-NN.

Requirements

• Please install OpenMS.
  • We recommend and tested using OpenMS version 3.4.0!
• If you want to run the python scripts, you need to install python 3.12 or higher or uv.

Usage

• On Microsoft Windows the applications can be run as standalone executables or as python scripts.
• Other operating systems are limited to the python scripts, please refer to CLI.md.

Graphical User Interface

We provide compiled binaries for Microsoft Windows that offer a graphical user interface. Please download the executables from releases.

Please make sure that the executable and the tmt18plex_default.ini file are in the same directory! You might also have to unblock the tmt18plex_default.ini file either via its Properties (right-click) or using PowerShell.

Commandline Interface

Please refer to CLI.md.

Running the Scripts for Multiple Files

If you want to run the scripts for multiple input files sequentially, please refer to MULTI.md.

Configuration

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 }

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.

TMT Resolution GUI Tool

You might also want to use the output of the Resolution GUI tool developed by Dina L. Bai, Tian Zhang et al. [1] as additional input for better quality control. Please refer to this repository for instructions: https://github.com/hgb-bin-proteomics/TMT_Resolution_GUI.

Contact

In case of questions please contact:

• micha.birklbauer@fh-hagenberg.at