MASIC (MS/MS Automated Selected Ion Chromatogram Generator)

MASIC generates selected ion chromatograms (SICs) for all of the parent ions chosen for fragmentation in an LC-MS/MS analysis. The SICs are generated using the LC-MS data, then each SIC is processed using a peak finding algorithm to characterize the chromatographic peaks, providing peak statistics including elution time of the peak apex, peak area, and peak signal/noise. The program can read instrument data in the following formats:

• Thermo .Raw files
• mzML files (see notes below)
• mzXML files
• mzData files
• .cdf/.mgf combo files

Results are saved both as flat files (.txt) and in an XML file that can be read using the accompanying graphical results browser, MASIC Browser. The browser provides a fast, graphical method for browsing the SICs identified by MASIC, allowing the user to sort and filter the SIC list as desired.

The application note describing MASIC is: Monroe ME et. al., Comput Biol Chem. 2008 Jun;32(3):215-217. More info is on PubMed

Downloads

Download a .zip file with the installer from: https://github.com/PNNL-Comp-Mass-Spec/MASIC/releases

The release page also includes a .zip file with MASIC_Console.exe

Installation

Double click file MASIC_Installer.exe file to install.
The program shortcut can be found at Start Menu -> Programs -> PAST Toolkit -> MASIC
The MASIC browser shortcut can be found at PAST Toolkit -> MASIC Browser

Console Version

The GUI version of MASIC, file MASIC.exe, includes a command line interface. To see the options, use MASIC.exe /?
See below for more information on the command line arguments.

Alternatively, use program MASIC_Console.exe, which is a command-line only version of MASIC. MASIC_Console.exe can be run on Linux using Mono

Custom SIC Values

You can optionally define custom values to instruct MASIC to create SICs of specific m/z values centered around a certain time point in the input file. You can define the custom values via three methods:

1. Using the Custom SIC Options tab in the GUI
2. Using a tab-delimited text file, which you select in the GUI or via the "CustomMZFile" setting in the "CustomSICValues" section of a MASIC XML parameter file
3. Using the "MZList", "ScanCenterList", and "ScanCommentList" settings in the "CustomSICValues" section of a MASIC XML parameter file

For all three methods, you can define three types of time modes:

• Absolute: absolute scan number
• Relative: relative time ranging from 0 (the first scan) to 1 (the last scan)
• AcquisitionTime: scan acquisition time, aka elution time (in minutes)

When defining custom SIC values, you must provide, at a minimum, the m/z value to search for. If no other values are provided, the default m/z search tolerance will be used (defined on the SIC Options tab in the GUI), and the entire dataset will be searched for the m/z value. You can optionally also define:

1. A custom m/z tolerance to use for each m/z

• If 0, the default SIC tolerance will be used

2. The central scan number or scan acquisition time you want the search to be centered around

• If 0, will be set to the mid-point of the dataset

3. The scan or time tolerance to search forward or backward from the central scan or time

• If 0, will use the default scan or time tolerance

4. A comment to associate with each custom m/z search value

If you use the GUI to define the Custom SIC values, then the column titles shown in the Custom SIC Values table will change based on the Tolerance Type defined.

If you define a tab-delimited text file from which to read the Custom SIC search details, the column headers present in the text file define the search tolerances to use. Files CustomMZList.txt and CustomMZList_AcqTime.txt, which are installed with MASIC, show the proper format for a Custom SIC Values file. At a minimum, the file must have a column titled "MZ" which specifies the custom m/z values to search for. If no other columns are present, a default tolerance is used for the m/z tolerance and the entire file will be searched for each custom m/z file. If you add other columns of data to the file, additional tolerances can be defined. Allowable column names are: MZToleranceDa, ScanCenter, ScanTolerance, and Comment

The data in columns ScanCenter and ScanTolerance can be absolute scan numbers, relative scan numbers, or acquisition time values. The mode to use can be defined in the GUI using the "Tolerance Type" option on the Custom SIC Options tab. Alternatively, if running MASIC from the Command Line, the mode to use must be defined using the "ScanType" setting in section "CustomSICValues" of an XML parameter file ("ScanType" can be "Absolute", "Relative", or "AcquisitionTime").

Instead of using column names ScanCenter and ScanTolerance you can alternatively use the column names ScanTime and TimeTolerance. When these columns are present, then the values in those columns will be treated as AcquisitionTime values, regardless of the global custom SIC Tolerance Type setting defined in the GUI or in the XML parameter file.

Output File Columns

_ScanStats.txt file columns

Column Name	Description
Dataset	Dataset name
ScanNumber	Scan number
ScanTime	Elution time (minutes)
ScanType	1 for MS1, 2 for MS2
TotalIonIntensity	Total Ion Intensity (TIC)
BasePeakIntensity	Base Peak Intensity (BPI)
BasePeakMZ	m/z value of the base peak
BasePeakSignalToNoiseRatio	S/N of the base peak
IonCount	Number of ions (after filtering)
IonCountRaw	Number of ions
ScanTypeName	HMS, HCD-HMSn, CID-MSn, etc.

_SICStats.txt file columns

Column Name	Description
Dataset	Dataset name
ParentIonIndex	Index
MZ	Parent ion m/z
SurveyScanNumber	Precursor scan number
FragScanNumber	MS2 scan number
OptimalPeakApexScanNumber	Scan number of the peak apex
PeakApexOverrideParentIonIndex	Index of the parent ion that this SIC inherits from; -1 if not inherited
CustomSICPeak	1 if a peak for a custom m/z search value
PeakScanStart	SIC peak start scan number
PeakScanEnd	SIC peak end scan number
PeakScanMaxIntensity	SIC peak center scan number
PeakMaxIntensity	Maximum intensity of the SIC peak
PeakSignalToNoiseRatio	Signal to noise ratio (S/N)
FWHMInScans	Full width half max, in scans
PeakArea	Area of the SIC peak
ParentIonIntensity	Intensity of the parent ion
PeakBaselineNoiseLevel	Baseline noise level surrounding the peak
PeakBaselineNoiseStDev	Baseline noise Standard Deviation
PeakBaselinePointsUsed	Number of points used to determine the baseline noise level
StatMomentsArea	Area, as computed using statistical moments
CenterOfMassScan	Central scan number, as determined via the center of mass (from statistical moments)
PeakStDev	Standard deviation, from statistical moments
PeakSkew	Skew, from statistical moments
PeakKSStat	KSStat, from statistical moments
StatMomentsDataCountUsed	Data points used by statistical moments calculations
InterferenceScore	Parent ion interference score, measuring the fraction of observed peaks in the parent ion isolation window that are from the precursor

Reporter Ions

Enable the "Generate Reporter Ion Stats" option to instruct MASIC to look for standard reporter ion masses and to save their observed intensities in file _ReporterIons.txt. Supported reporter ion modes are shown in the following table The integer value corresponds to the number that appears in the MASIC settings file, e.g. <item key="ReporterIonMassMode" value="16" /> for TMT 11

Integer Value	Name	Description	Ions (m/z values)
1	iTraq	Original 4-plex iTRAQ	114.1112, 115.1083, 116.1116, and 117.115
2	ITraq ETD	iTRAQ ETD tags	101.107, 102.104, and 104.1107
5	ITraq 8 (high res)	High resolution eight-plex iTRAQ	113.107873, 114.111228, 115.108263, 116.111618, 117.114973, 118.112008, 119.115363, and 121.122072
6	ITraq 8 (low res)	Low resolution eight-plex iTRAQ	Same ions as Mode 5, but corrects for contamination from the immonium ion at 120.08131
3	TMT 2	TMT duplex	126.1283 and 127.1316
4	TMT 6	6-plex TMT	126.1283, 127.1316, 128.135, 129.1383, 130.1417, and 131.1387
10	TMT 10	10-plex TMT	126.127726, 127.124761, 127.131081, 128.128116, 128.134436, 129.131471, 129.13779, 130.134825, 130.141145, and 131.138180
16	TMT 11	11-plex TMT	126.127726, 127.124761, 127.131081, 128.128116, 128.134436, 129.131471, 129.13779, 130.134825, 130.141145, 131.138180, and 131.144499
18	TMT 16	16-plex TMT (aka TMTpro)	126.127726, 127.124761, 127.131081, 128.128116, 128.134436, 129.131471, 129.137791, 130.134826, 130.141146, 131.138181, 131.144501, 132.141536, 132.147856, 133.144891, 133.151211, 134.148246
20	TMT 18	18-plex TMT (aka TMTpro 18plex)	126.127726, 127.124761, 127.131081, 128.128116, 128.134436, 129.131471, 129.137791, 130.134826, 130.141146, 131.138181, 131.144501, 132.141536, 132.147856, 133.144891, 133.151211, 134.148246, 134.154566, 135.151601
21	TMT 32	32-plex TMT (aka TMTpro 32plex)	126.127726, 127.124761, 127.131081, 128.128116, 128.134436, 129.131471, 129.137791, 130.134826, 130.141146, 131.138181, 131.144501, 132.141536, 132.147856, 133.144891, 133.151211, 134.148246, 127.134003, 128.131038, 128.137358, 129.134393, 129.140713, 130.137748, 130.144068, 131.141103, 131.147423, 132.144458, 132.150778, 133.147813, 133.154133, 134.151171, 134.157491, 135.154526
22	TMT 35	35-plex TMT (aka TMTpro 35plex)	126.127726, 127.124761, 127.131081, 128.128116, 128.134436, 129.131471, 129.137791, 130.134826, 130.141146, 131.138181, 131.144501, 132.141536, 132.147856, 133.144891, 133.151211, 134.148246, 134.154566, 135.151601, 127.134003, 128.131038, 128.137358, 129.134393, 129.140713, 130.137748, 130.144068, 131.141103, 131.147423, 132.144458, 132.150778, 133.147813, 133.154133, 134.151171, 134.157491, 135.154526, 135.160846
7	PCGalnaz	Custom reporter ions for Josh Alfaro	300.13 and 503.21 m/z
8	HemeCFragment	Custom reporter ions for Eric Merkley	616.1767 and 617.1845
9	LycAcetFragment	Custom reporter ions for Ernesto Nakayasu	126.09134 and 127.094695
11	OGlcNAc	O-GlcNAc	204.0872, 300.13079, and 503.21017
12	Fracking Amine 20160217	Product ions associated with a Fracking Fluid amine	157.089, 170.097, and 234.059
13	FSFACustomCarbonyl	Custom product ions from Chengdong Xu	171.104, 236.074, and 257.088
14	FSFACustomCarboxylic	Custom product ions from Chengdong Xu	171.104, 234.058, and 336.174
15	FSFACustomHydroxyl	Custom product ions from Chengdong Xu	151.063 and 166.087
17	Acetylation	Peptides with acetylated lysine residues	126.09134 and 143.11789
19	NativeOGlcNAc	Native O-GlcNAc	126.055, 138.055, 144.065, 168.066, 186.076, 204.087, and 366.14

TMT 32 and TMT 35 notes:

• 32-plex TMT is a combination of non-deuterated 16-plex TMT and a deuterated 16-plex version, which requires a resolution of over 75,000 to resolve the reporter ions
• 35-plex TMT is a combination of non-deuterated 18-plex TMT and a deuterated 17-plex version, which requires a resolution of over 75,000 to resolve the reporter ions

For datasets with low resolution MS2 spectra but high resolution MS3 spectra, MASIC will copy the reporter ion abundances from the MS3 spectra to the MS2 spectra if the median of the reporter ion abundances for a given scan is larger in the MS3 spectrum vs. the parent MS2 spectrum. This behavior can be adjusted using options UseMS3ReporterIonsForParentMS2Spectra and AlwaysUseMS3ReporterIonsForParents in a MASIC parameter file:

• When UseMS3ReporterIonsForParentMS2Spectra is True and AlwaysUseMS3ReporterIonsForParents is False
  • Only copy from MS3 to MS2 if the median intensity is larger for the MS3 reporter ions
  • This is the default behavior
• When UseMS3ReporterIonsForParentMS2Spectra is True and AlwaysUseMS3ReporterIonsForParents is True
  • Always copy reporter ion intensities from MS3 spectra to the parent MS2 spectra
• When UseMS3ReporterIonsForParentMS2Spectra is False
  • Never copy reporter ion intensities from MS3 spectra to the parent MS2 spectra

MRM Data

When processing Thermo-Finnigan MRM data files, a file named _MRMSettings.txt will be created listing the parent and daughter m/z values monitored via selected reaction monitoring. You can optionally export detailed MRM intensity data using the "MRM Data List" and/or "MRM Intensity Crosstab" options. The MRM Data List file is a long, narrow file with only a few columns: Scan, MRM_Parent_MZ, MRM_Daughter_MZ, and MRM_Daughter_Intensity. In contrast, the MRM Intensity Crosstab file is a wide, rectangular file that presents the data in a crosstab format (aka PivotTable). Here, each column in the crosstab corresponds to the intensity values over time for a given parent m/z to daughter m/z transition being monitored.

If you convert a MRM file to the .mzML format using the MSConvert utility in ProteoWizard, you need to use command line switches --srmAsSpectra and --simAsSpectra. Otherwise, the PSI_Interface utility which we use to read .mzML files cannot load the mass spectra data. Example command line:

msconvert.exe --32 --mzML --srmAsSpectra --simAsSpectra DatasetName.raw

IonCountRaw vs. IonCount

In the ScanStats file, IonCountRaw is the number MASIC m/z values present in each spectrum. IonCount is the number of points that MASIC keeps in memory and examines when creating SICs. For LTQ-FT data with scans containing lots of high resolution data, MASIC compresses the data a bit to combine m/z values that are within ~0.05 m/z (thus, combine 1000.05 and 1000.052 as simply 1000.051). Additionally, for scans with a lot of low quality, low intensity data, MASIC discards the low intensity data. The IonCount value would let you see which scans MASIC is discarding some data, for whatever reason.

.mzML Support

Although MASIC can read .mzML files, when possible, you should process Thermo raw files directly with MASIC. The reason is that the m/z and intensity values obtained using ThermoRawFileReader.dll and ThermoFisher.CommonCore.Data.dll more accurately represent the true data, in particular for centroided data obtained from a profile mode scan.

The following observations are based on comparing results from .mzML files created using the following options with a Thermo .raw file that has profile mode MS1 and MS2 scans

Profile mode .mzML file

msconvert.exe --32 --mzML DatasetName.raw

Centroid mode .mzML file

msconvert.exe --32 --mzML --filter "peakPicking true 1-" DatasetName.raw

Peak intensities (and thus areas) reported by MASIC

• .mzML profile mode data has intensities and areas nearly identical to Thermo .raw files
• .mzML centroid mode data has intensities and areas ~2.5 fold smaller than Thermo .raw files

Reporter Ion Intensities

• .mzML profile mode data has identical reporter ion intensities vs. Thermo .raw files
• .mzML centroid mode data has similar reporter ion intensities vs. Thermo .raw files (agreement within 2%)

Precursor Ion Interference Scores

• .mzML profile mode data has similar interference scores only for higher abundance precursors; for lower abundance precursors, the centroiding algorithm does not perform well (m/z values deviate from their true values), and thus the interference scores do not correlate well with Thermo .raw file based scores
• .mzML centroid mode data has identical interference scores as Thermo .raw files

Based on the above observations, when reading .mzML files, centroided MS1 spectra work better for interference score calculations, but intensities are 2.5 fold smaller. In contrast, profile mode MS2 spectra more accurately represent reporter ion intensities.

MSConvert supports creating files with centroided MS1 spectra and profile mode MS2 spectra:

msconvert.exe --32 --mzML --filter "peakPicking true 1" DatasetName.raw

Plots

The PlotOptions section in the MASIC parameter file has options for instructing MASIC to create various plots. When enabled, the following plots are created:

Plot Title	Filename	Description
Peak area histogram	Dataset_PeakAreaHistogram.png	Histogram of selected ion chromatogram (SIC) peak areas. Areas are log-10 transformed.
Peak width histogram	Dataset_PeakWidthHistogram.png	Histogram of peak widths (FWHM) of selected ion chromatogram peaks (in seconds).
Reporter ion observation rate	Dataset_RepIonObsRate.png	Bar chart showing the percentage of MS/MS spectra for which a reporter ion was observed, showing a separate bar for each channel.
Reporter ion observation rate, top 80%	Dataset_RepIonObsRateHighAbundance.png	Bar chart showing the percentage of MS/MS spectra for which a reporter ion was observed. Uses data from the top 80% of spectra, as sorted by SIC peak area (% is adjustable).
Reporter ion intensity stats, top 80%	Dataset_RepIonStatsHighAbundance.png	Box and whiskers plot showing the distribution of reporter ion intensities, by channel. Uses data from the top 80% of spectra, as sorted by SIC peak area (% is adjustable).

The reporter ion based plots are only created if reporter ions were searched for.

Example plots are visible on GitHub

• TMT Example
• Incomplete TMT labeling example
• QExactive Example
• QExactive HFX Example
• QExactive HF Example

By default, plots are created using OxyPlot, which only works on Windows

To create plots with Python instead of OxyPlot, set PlotWithPython to True in the parameter file

• For example, see TMT11_LTQ-FT_10ppm_ReporterTol0.003Da_2017-03-17.xml

On Windows, MASIC looks for python.exe in directories that start with "Python3" or "Python 3", searching below:

• C:\Program Files
• C:\Program Files (x86)
• C:\Users\Username\AppData\Local\Programs
• C:\ProgramData\Anaconda3
• C:\

On Linux, assumes Python is at /usr/bin/python3

Python plotting requires that three libraries be installed

• numpy
• matplotlib
• pandas

For Python library installation options, see the Python_Setup.txt file on GitHub

• https://github.com/PNNL-Comp-Mass-Spec/MASIC/blob/master/Python/Python_Setup.txt

The plot data can optionally be saved as tab-delimited text files

• Enable by setting SaveHistogramData and/or SaveReporterIonObservationRateData to True in the parameter file

Plots can be created from existing MASIC results by providing the _SICStats.txt or _ScanStats.txt file name as the input file

Command Line Interface

Both MASIC.exe and MASIC_Console.exe include the same command line interface.

MASIC.exe
 /I:InputFilePath [/O:OutputDirectoryPath]
 [/P:ParamFilePath] [/D:DatasetID or DatasetLookupFilePath]
 [/S:[MaxLevel]] [/A:AlternateOutputDirectoryPath] [/R]
 [/L:[LogFilePath]] [/LogDir:LogDirPath] [/SF:StatusFileName] 
 [/CreateParamFile:FileName.xml] [/Q]

The input file path can contain the wildcard character *

• It will typically point to an instrument data file (e.g. Thermo .raw file)

If the input file name ends with _SICstats.txt, MASIC will create plots using existing results

• If a _ReporterIons.txt file is also present, the Reporter Ion Observation Rate plots will also be made

The output directory name is optional. If omitted, the output files will be created in the same directory as the input file. If included, a subdirectory will be created with the name OutputDirectoryName.

The parameter file switch /P is optional. If supplied, it should point to a valid MASIC XML parameter file. If omitted, defaults are used.

• Create a parameter file for MASIC using the MASIC GUI
• Alternatively, download a file from GitHub

The /D switch can be used to specify the Dataset ID of the input file; if omitted, 0 will be used

Alternatively, a lookup file can be specified with the /D switch (useful if processing multiple files using * or /S). The lookup file is a comma, space, or tab delimited file with two columns:
Dataset Name and Dataset ID

Use /S to process all valid files in the input directory and subdirectories. Include a number after /S (like /S:2) to limit the level of subdirectories to examine.

• When using /S, you can redirect the output of the results using /A.
• When using /S, you can use /R to re-create the input directory hierarchy in the alternate output directory (if defined).

Use /L to specify that a log file should be created. Use /L:LogFilePath to specify the name (or full path) for the log file.

Use /SF to specify the name to use for the MASIC Status file (default is MasicStatus.xml)

Use /CreateParamFile to create an example parameter file named MASIC_ExampleSettings.xml

• Include a colon and a filename to customize the filename

The optional /Q switch will prevent the progress window from being shown (only applicable to the GUI version, MASIC.exe)

Compiling MASIC

Steps for compiling MASIC from source code

• Download the source code by cloning the MASIC repository from GitHub
• Open with Visual Studio
• Use the NuGet Package Manager GUI to remove the reference to local package ThermoRawFileReader
  • Tools, NuGet Package Manager, Manager NuGet Packages for Solution
  • Select the Installed tab
  • Click ThermoRawFileReader
  • Click Uninstall
• Use the NuGet Package Manager GUI to add a reference to OpenMcdf.Extensions
  • Tools, NuGet Package Manager, Manager NuGet Packages for Solution
  • Select the Browse tab
  • Search for OpenMcdf.Extensions
  • Select MASIC_Console.csproj and/or MASIC.csproj project
  • Click Install
• Download the latest release of the ThermoRawFileReader from GitHub
• Add a reference to the following five DLLs, available in the ThermoRawFileReader release .zip file
  • ThermoRawFileReader.dll
  • ThermoFisher.CommonCore.Data.dll
  • ThermoFisher.CommonCore.MassPrecisionEstimator.dll
  • ThermoFisher.CommonCore.RawFileReader.dll
  • ThermoFisher.CommonCore.BackgroundSubtraction.dll
• Build the project

Contacts

Written by Matthew Monroe for the Department of Energy (PNNL, Richland, WA)
Copyright 2019, Battelle Memorial Institute. All Rights Reserved.
E-mail: [email protected] or [email protected]
Website: https://github.com/PNNL-Comp-Mass-Spec/ or https://www.pnnl.gov/integrative-omics

License

MASIC is licensed under the 2-Clause BSD License; you may not use this program except in compliance with the License. You may obtain a copy of the License at https://opensource.org/licenses/BSD-2-Clause

Copyright 2018 Battelle Memorial Institute

RawFileReader reading tool. Copyright © 2016 by Thermo Fisher Scientific, Inc. All rights reserved.