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<div class="title">IsobaricAnalyzer </div> </div>
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<div class="textblock"><p>Extracts and normalizes isobaric labeling information from an MS experiment.</p>
<center> <table class="doxtable">
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<td align="center" bgcolor="#EBEBEB">pot. predecessor tools </td><td valign="middle" rowspan="3"><img class="formulaInl" alt="$ \longrightarrow $" src="form_91.png"/> IsobaricAnalyzer <img class="formulaInl" alt="$ \longrightarrow $" src="form_91.png"/> </td><td align="center" bgcolor="#EBEBEB">pot. successor tools </td></tr>
<tr>
<td valign="middle" align="center" rowspan="1"><a class="el" href="TOPP_FileConverter.html">FileConverter</a> </td><td valign="middle" align="center" rowspan="2"><a class="el" href="TOPP_IDMapper.html">IDMapper</a> </td></tr>
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<td valign="middle" align="center" rowspan="1"><a class="el" href="TOPP_FileFilter.html">FileFilter</a> </td></tr>
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</center><p>Extract the isobaric reporter ion intensities (currently iTRAQ 4plex and 8plex and TMT 6plex are supported) from raw MS2 data, does isotope corrections and stores the resulting quantitation as consensusXML, where each consensus centroid corresponds to one isobaric MS2 scan (e.g., HCD). The position of the centroid is the precursor position, its sub-elements are the channels (thus having m/z's of 113-121 for iTRAQ 126-131 for TMT respectively).</p>
<p>Isotope correction is done using non-negative least squares (NNLS), i.e.,</p>
<p>Minimize ||Ax - b||, subject to x >= 0, where b is the vector of observed reporter intensities (with 'contaminating' isotope species), A is a correction matrix (as supplied by the manufacturer AB Sciex) and x is the desired vector of corrected (real) reporter intensities. Other software solves this problem using an inverse matrix multiplication, but this can yield entries in x which are negative. In a real sample, this solution cannot possibly be true, so usually negative values (= negative reporter intensities) are set to 0. However, a negative result usually means, that noise was not accounted for thus we use NNLS to get a non-negative solution, without the need to truncate negative values. In (the usual) case that inverse matrix multiplication yields only positive values, our NNLS will give the exact same optimal solution.</p>
<p>The correction matrices can be found (and changed) in the INI file. However, these matrices for both 4plex and 8plex iTRAQ are now stable, and every kit delivered should have the same isotope correction values. Thus, there should be no need to change them, but feel free to compare the values in the INI file with your kit's Certificate. For TMT 6plex the values have to be adapted for each kit.</p>
<p>After this quantitation step, you might want to annotate the consensus elements with the respective identifications, obtained from an identification pipeline. Note that quantification is solely on peptide level at this stage. In order to obtain protein quantifications, you can try <a class="el" href="TOPP_TextExporter.html">TextExporter</a> to obtain a simple text format which you can feed to other software tools (e.g., R), or you can try <a class="el" href="TOPP_ProteinQuantifier.html">ProteinQuantifier</a>.</p>
<p><b>The command line parameters of this tool are:</b> </p>
<pre class="fragment">
IsobaricAnalyzer -- Calculates isobaric quantitative values for peptides
Version: 1.11.1 Nov 14 2013, 11:18:15, Revision: 11976
Usage:
IsobaricAnalyzer <options>
This tool has algoritm parameters that are not shown here! Please check the ini file for a detailed descripti
on or use the --helphelp option.
Options (mandatory options marked with '*'):
-type <mode> Isobaric Quantitation method used in the experiment. (default: 'itraq4plex' valid: 'itra
q4plex', 'itraq8plex', 'tmt6plex')
-in <file>* Input raw/picked data file (valid formats: 'mzML')
-out <file>* Output consensusXML file with quantitative information (valid formats: 'consensusXML')
Common TOPP options:
-ini <file> Use the given TOPP INI file
-threads <n> Sets the number of threads allowed to be used by the TOPP tool (default: '1')
-write_ini <file> Writes the default configuration file
-id_pool <file> ID pool file to DocumentID's for all generated output files. Disabled by default. (Set
to 'main' to use /Users/aiche/dev/openms/openms-1.11.1/share/OpenMS/IDPool/IDPool.txt)
--help Shows options
--helphelp Shows all options (including advanced)
The following configuration subsections are valid:
- extraction Parameters for the channel extraction.
- itraq4plex Algorithm parameters for itraq4plex
- itraq8plex Algorithm parameters for itraq8plex
- quantification Parameters for the peptide quantification.
- tmt6plex Algorithm parameters for tmt6plex
You can write an example INI file using the '-write_ini' option.
Documentation of subsection parameters can be found in the doxygen documentation or the INIFileEditor.
Have a look at the OpenMS documentation for more information.
</pre><p> <b>INI file documentation of this tool:</b> <div class="ini_global">
<div class="legend">
<b>Legend:</b><br>
<div class="item item_required">required parameter</div>
<div class="item item_advanced">advanced parameter</div>
</div>
<div class="node"><span class="node_name">+IsobaricAnalyzer</span><span class="node_description">Calculates isobaric quantitative values for peptides</span></div>
<div class="item item_advanced"><span class="item_name" style="padding-left:16px;">version</span><span class="item_value">1.11.1</span>
<span class="item_description">Version of the tool that generated this parameters file.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="node"><span class="node_name">++1</span><span class="node_description">Instance '1' section for 'IsobaricAnalyzer'</span></div>
<div class="item"><span class="item_name" style="padding-left:24px;">type</span><span class="item_value">itraq4plex</span>
<span class="item_description">Isobaric Quantitation method used in the experiment.</span><span class="item_tags"></span><span class="item_restrictions">itraq4plex,itraq8plex,tmt6plex</span></div> <div class="item"><span class="item_name item_required" style="padding-left:24px;">in</span><span class="item_value"></span>
<span class="item_description">input raw/picked data file </span><span class="item_tags">input file</span><span class="item_restrictions">*.mzML</span></div> <div class="item"><span class="item_name item_required" style="padding-left:24px;">out</span><span class="item_value"></span>
<span class="item_description">output consensusXML file with quantitative information</span><span class="item_tags">output file</span><span class="item_restrictions">*.consensusXML</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:24px;">log</span><span class="item_value"></span>
<span class="item_description">Name of log file (created only when specified)</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:24px;">debug</span><span class="item_value">0</span>
<span class="item_description">Sets the debug level</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:24px;">threads</span><span class="item_value">1</span>
<span class="item_description">Sets the number of threads allowed to be used by the TOPP tool</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:24px;">no_progress</span><span class="item_value">false</span>
<span class="item_description">Disables progress logging to command line</span><span class="item_tags"></span><span class="item_restrictions">true,false</span></div> <div class="item"><span class="item_name" style="padding-left:24px;">id_pool</span><span class="item_value"></span>
<span class="item_description">ID pool file to DocumentID's for all generated output files. Disabled by default. (Set to 'main' to use /Users/aiche/dev/openms/openms-1.11.1/share/OpenMS/IDPool/IDPool.txt)</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:24px;">test</span><span class="item_value">false</span>
<span class="item_description">Enables the test mode (needed for internal use only)</span><span class="item_tags"></span><span class="item_restrictions">true,false</span></div> <div class="node"><span class="node_name">+++extraction</span><span class="node_description">Parameters for the channel extraction.</span></div>
<div class="item"><span class="item_name" style="padding-left:32px;">select_activation</span><span class="item_value">High-energy collision-induced dissociation</span>
<span class="item_description">Operate only on MSn scans where any of its precursors features a certain activation method (e.g., usually HCD for iTRAQ). Set to empty string if you want to disable filtering.</span><span class="item_tags"></span><span class="item_restrictions">Collision-induced dissociation,Post-source decay,Plasma desorption,Surface-induced dissociation,Blackbody infrared radiative dissociation,Electron capture dissociation,Infrared multiphoton dissociation,Sustained off-resonance irradiation,High-energy collision-induced dissociation,Low-energy collision-induced dissociation,Photodissociation,Electron transfer dissociation,</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">reporter_mass_shift</span><span class="item_value">0.1</span>
<span class="item_description">Allowed shift (left to right) in Da from the expected position.</span><span class="item_tags"></span><span class="item_restrictions">1e-08:0.5</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">min_precursor_intensity</span><span class="item_value">1</span>
<span class="item_description">Minimum intensity of the precursor to be extracted. MS/MS scans having a precursor with a lower intensity will not be considered for quantitation.</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">keep_unannotated_precursor</span><span class="item_value">true</span>
<span class="item_description">Flag if precursor with missing intensity value or missing precursor spectrum should be included or not.</span><span class="item_tags"></span><span class="item_restrictions">true,false</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">min_reporter_intensity</span><span class="item_value">0</span>
<span class="item_description">Minimum intenesity of the individual reporter ions to be used extracted.</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">discard_low_intensity_quantifications</span><span class="item_value">false</span>
<span class="item_description">Remove all reporter intensities if a single reporter is below the threshold given in min_reporter_intensity.</span><span class="item_tags"></span><span class="item_restrictions">true,false</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">min_precursor_purity</span><span class="item_value">0</span>
<span class="item_description">Minimum fraction of the total intensity in the isolation window of the precursor spectrum attributable to the selected precursor.</span><span class="item_tags"></span><span class="item_restrictions">0:1</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">precursor_isotope_deviation</span><span class="item_value">0.02</span>
<span class="item_description">Maximum allowed deviation between theoretical and observed isotopic peaks of the precursor peak in the isolation window to be counted as part of the precursor.</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="node"><span class="node_name">+++itraq4plex</span><span class="node_description">Algorithm parameters for itraq4plex</span></div>
<div class="item"><span class="item_name" style="padding-left:32px;">channel_114_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 114 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_115_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 115 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_116_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 116 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_117_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 117 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">reference_channel</span><span class="item_value">114</span>
<span class="item_description">Number of the reference channel (114-117).</span><span class="item_tags"></span><span class="item_restrictions">114:117</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">correction_matrix</span><span class="item_value">[0.0/1.0/5.9/0.2, 0.0/2.0/5.6/0.1, 0.0/3.0/4.5/0.1, 0.1/4.0/3.5/0.1]</span>
<span class="item_description">Override default values (see Documentation); use the following format: <-2Da>/<-1Da>/<+1Da>/<+2Da> ; e.g. '0/0.3/4/0' , '0.1/0.3/3/0.2'</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="node"><span class="node_name">+++itraq8plex</span><span class="node_description">Algorithm parameters for itraq8plex</span></div>
<div class="item"><span class="item_name" style="padding-left:32px;">channel_113_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 113 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_114_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 114 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_115_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 115 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_116_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 116 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_117_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 117 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_118_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 118 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_119_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 119 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_121_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 121 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">reference_channel</span><span class="item_value">113</span>
<span class="item_description">Number of the reference channel (113-121). Please note that 120 is not valid.</span><span class="item_tags"></span><span class="item_restrictions">113:121</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">correction_matrix</span><span class="item_value">[0.00/0.00/6.89/0.22, 0.00/0.94/5.90/0.16, 0.00/1.88/4.90/0.10, 0.00/2.82/3.90/0.07, 0.06/3.77/2.99/0.00, 0.09/4.71/1.88/0.00, 0.14/5.66/0.87/0.00, 0.27/7.44/0.18/0.00]</span>
<span class="item_description">Override default values (see Documentation); use the following format: <-2Da>/<-1Da>/<+1Da>/<+2Da> ; e.g. '0/0.3/4/0' , '0.1/0.3/3/0.2'</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="node"><span class="node_name">+++quantification</span><span class="node_description">Parameters for the peptide quantification.</span></div>
<div class="item"><span class="item_name" style="padding-left:32px;">isotope_correction</span><span class="item_value">true</span>
<span class="item_description">Enable isotope correction (highly recommended). Note that you need to provide a correct isotope correction matrix otherwise the tool will fail or produce invalid results.</span><span class="item_tags"></span><span class="item_restrictions">true,false</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">normalization</span><span class="item_value">false</span>
<span class="item_description">Enable normalization of channel intensities with respect to the reference channel. The normalization is done by using the Median of Ratios (every channel / Reference). Also the ratio of medians (from any channel and reference) is provided as control measure!</span><span class="item_tags"></span><span class="item_restrictions">true,false</span></div> <div class="node"><span class="node_name">+++tmt6plex</span><span class="node_description">Algorithm parameters for tmt6plex</span></div>
<div class="item"><span class="item_name" style="padding-left:32px;">channel_126_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 126 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_127_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 127 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_128_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 128 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_129_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 129 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_130_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 130 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">channel_131_description</span><span class="item_value"></span>
<span class="item_description">Description for the content of the 131 channel.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">reference_channel</span><span class="item_value">126</span>
<span class="item_description">Number of the reference channel (126-131).</span><span class="item_tags"></span><span class="item_restrictions">126:131</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">correction_matrix</span><span class="item_value">[0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0]</span>
<span class="item_description">Override default values (see Documentation); use the following format: <-2Da>/<-1Da>/<+1Da>/<+2Da> ; e.g. '0/0.3/4/0' , '0.1/0.3/3/0.2'</span><span class="item_tags"></span><span class="item_restrictions"> </span></div></div>
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