The Interference Detector library implements an algorithm for quantifying the homogeneity of species isolated for fragmentation in LC-MS/MS analyses using data directed acquisition (DDA). It works with Thermo .Raw files and _isos.csv files from DeconTools. The inteference score computed for each parent ion is the fraction of ions in the isolation window that are from the precursor (weighted by intensity).
In DDA analyses, compounds (e.g. peptides) are isolated then bombarded with extra energy to cause the compound to break apart into components (fragments). Ideally a single ion is isolated for fragmentation, but in complex samples, multiple species may be isolated simultaneously.
The InterferenceDetector class examines the parent MS1 spectrum of each fragmented ion, looking for other ions that were included in the isolation window. It computes an interference score for each parent ion, writing the results to a new SQLite table.
The DMS Analysis Manager uses InterDetect.dll when processing iTRAQ or TMT data to compute interference scores.
The IDM_Console.exe program can be used to manually process a SQLite file.
IDM_Console.exe
InputFile.db3
[/Tol:PrecursorTolerancePPM]
[/CSTol:ChargeStateEstimationTolerance]
[/KeepTemp]
The input file is a SQLite database with table T_MSMS_Raw_Files
and optionally either T_Results_Metadata_Typed or T_Results_Metadata
Use /Tol to specify the PPM tolerance when looking for the precursor ion in the isolation window The default is +/-15 PPM
Use /CSTol to specify the m/z tolerance when guesstimating the charge The default is +/-0.01 m/z
Use /KeepTemp to not delete the temporary precursor info file, prec_info_temp.txt
A SQLite file with the following tables.
T_MSMS_Raw_Files
| Column | Description |
|---|---|
| Dataset_ID | Dataset ID |
| Dataset | Dataset Name |
| Folder | Dataset Folder Path |
The program assumes that datasets in T_MSMS_Raw_Files are Thermo datasets
and will therefore append extension .raw to the dataset names when determining
the dataset file path.
Optionally a table with DeconTools job info. This info can be
in either T_Results_Metadata_Typed or T_Results_Metadata
| Column | Description |
|---|---|
| Tool | Analysis Tool Name |
| Dataset_ID | Dataset ID |
| Dataset | Dataset Name |
| Folder | Dataset Folder Path |
The program looks for rows in this table where the tool name starts with Decon.
It is not a fatal error if this table is missing, or if the table has no DeconTools jobs. If the file does exist, it will be used to determine the charge state of parent ions listed in the .Raw file as having a charge of 0 (unknown charge). The tolerance used when finding matching ions in the _isos.csv file is +/-0.005 m/z
The _isos.csv file is a comma separated file created by DeconTools. Required columns:
| Column | Description |
|---|---|
| scan_num | Scan number |
| mz | Ion m/z |
| charge | Charge state |
| abundance | Ion abundance |
The InterferenceDetector class opens the Results.db3 file and determines
the datasets to process using tables T_MSMS_Raw_Files and T_Results_Metadata_Typed.
For each dataset, it copies the thermo .Raw file locally, then reads the data
using the ThermoRawFileReader. It also looks for the _isos.csv file and
caches the data if found.
For each ion chosen for fragmentation, the ions in the region of the precursor ion (plus or minus the isolation width) are examined. If the charge state was not determined by the Thermo acquisition software or by DeconTools, is is estimated using the ChargeStateGuesstimator method. If the charge cannot be guesstimated, the interference score for the ion will be 0.
For precursor ions with a known charge, the algorithm computes the m/z difference (in ppm) between a given ion and the precursor ion. If this difference corresponds to an expected difference for the given charge state (+/- 15 ppm), the ion is considered to have arisen from the precursor.
The algorithm computes the sum of the intensity of all of the ions in the isolation window,
along with a sum of the intensity of all of the precursor-associated ions in the window,
then computes the interference score as
interferenceScore = intensitySumPrecursorIons / intensitySumAllPeaks
Example calculations
| Precursor Ions Intensity sum | All Ions Intensity Sum | Inteference Score |
|---|---|---|
| 22102 | 31812 | 0.695 |
| 8593334 | 8936750 | 0.962 |
| 201366 | 410589 | 0.490 |
| 281669 | 320436 | 0.879 |
An Inteference score of 1 means that all of the peaks in the isolation window were from the precursor ion.
The program will create (or replace) table T_Precursor_Interference with columns:
| Column | Description |
|---|---|
| Dataset_ID | Dataset ID |
| ScanNumber | MS/MS scan number |
| PrecursorScan | Precursor (MS1) scan number |
| ParentMZ | Parent ion m/z |
| ChargeState | Parent ion charge state |
| IsoWidth | Isolation width |
| Interference | Interference score; larger is better, with a max of 1 and minimum of 0 |
| PreIntensity | Parent ion abundance in the precursor scan |
| IonCollectionTime | Ion Injection Time (ms) |
This data is also written to file prec_info_temp.txt ,though that file
is deleted after the information is successfully added to the SQLite database.
Written by Josh Aldrich for the Department of Energy (PNNL, Richland, WA)
E-mail: [email protected]
Website: https://github.com/PNNL-Comp-Mass-Spec/ or https://www.pnnl.gov/integrative-omics
The Interference Detector library is licensed under the Apache License, Version 2.0; you may not use this file except in compliance with the License. You may obtain a copy of the License at https://opensource.org/licenses/Apache-2.0