1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
|
<HTML>
<HEAD>
<TITLE>SILACAnalyzer</TITLE>
<LINK HREF="doxygen.css" REL="stylesheet" TYPE="text/css">
<LINK HREF="style_ini.css" REL="stylesheet" TYPE="text/css">
</HEAD>
<BODY BGCOLOR="#FFFFFF">
<A href="index.html">Home</A> ·
<A href="classes.html">Classes</A> ·
<A href="annotated.html">Annotated Classes</A> ·
<A href="modules.html">Modules</A> ·
<A href="functions_func.html">Members</A> ·
<A href="namespaces.html">Namespaces</A> ·
<A href="pages.html">Related Pages</A>
<HR style="height:1px; border:none; border-top:1px solid #c0c0c0;">
<!-- Generated by Doxygen 1.8.5 -->
</div><!-- top -->
<div class="header">
<div class="headertitle">
<div class="title">SILACAnalyzer </div> </div>
</div><!--header-->
<div class="contents">
<div class="textblock"><p>Identifies peptide pairs in LC-MS data and determines their relative abundance.</p>
<center> <table class="doxtable">
<tr>
<td align="center" bgcolor="#EBEBEB">pot. predecessor tools </td><td valign="middle" rowspan="3"><img class="formulaInl" alt="$ \longrightarrow $" src="form_91.png"/> SILACAnalyzer <img class="formulaInl" alt="$ \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>
<tr>
<td valign="middle" align="center" rowspan="1"><a class="el" href="TOPP_FileFilter.html">FileFilter</a> </td></tr>
</table>
</center><p>SILACAnalyzer is a tool for the fully automated analysis of quantitative proteomics data. It identifies pairs of isotopic envelopes with fixed m/z separation. It requires no prior sequence identification of the peptides. In what follows we first explain the algorithm and then discuss the tuning of its parameters.</p>
<p><b>Algorithm</b></p>
<p>The algorithm is divided into three parts: filtering, clustering and linear fitting, see Fig. (d), (e) and (f). In the following discussion let us consider a particular mass spectrum at retention time 1350 s, see Fig. (a). It contains a peptide of mass 1492 Da and its 6 Da heavier labelled counterpart. Both are doubly charged in this instance. Their isotopic envelopes therefore appear at 746 and 749 in the spectrum. The isotopic peaks within each envelope are separated by 0.5. The spectrum was recorded at finite intervals. In order to read accurate intensities at arbitrary m/z we spline-fit over the data, see Fig. (b).</p>
<p>We would like to search for such peptide pairs in our LC-MS data set. As a warm-up let us consider a standard intensity cut-off filter, see Fig. (c). Scanning through the entire m/z range (red dot) only data points with intensities above a certain threshold pass the filter. Unlike such a local filter, the filter used in our algorithm takes intensities at a range of m/z positions into account, see Fig. (d). A data point (red dot) passes if</p>
<ul>
<li>all six intensities at m/z, m/z+0.5, m/z+1, m/z+3, m/z+3.5 and m/z+4 lie above a certain threshold,</li>
<li>the intensity profiles in neighbourhoods around all six m/z positions show a good correlation and</li>
<li>the relative intensity ratios within a peptide agree up to a factor with the ratios of a theoretic averagine model.</li>
</ul>
<p>Let us now filter not only a single spectrum but all spectra in our data set. Data points that pass the filter form clusters in the t-m/z plane, see Fig. (e). Each cluster corresponds to the mono-isotopic mass trace of the lightest peptide of a SILAC pattern. We now use hierarchical clustering methods to assign each data point to a specific cluster. The optimum number of clusters is determined by maximizing the silhouette width of the partitioning. Each data point in a cluster corresponds to three pairs of intensities (at [m/z, m/z+3], [m/z+0.5, m/z+3.5] and [m/z+1, m/z+4]). A plot of all intensity pairs in a cluster shows a clear linear correlation, see Fig. (f). Using linear regression we can determine the relative amounts of labelled and unlabelled peptides in the sample.</p>
<div class="image">
<img src="SILACAnalyzer_algorithm.png" alt="SILACAnalyzer_algorithm.png"/>
</div>
<p><b>The command line parameters of this tool are:</b> </p>
<pre class="fragment">
SILACAnalyzer -- Determination of peak ratios in LC-MS data
Version: 1.11.1 Nov 14 2013, 11:18:15, Revision: 11976
Usage:
SILACAnalyzer <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 '*'):
-in <file>* Raw LC-MS data to be analyzed. (Profile data required. Will not work with centroided
data!) (valid formats: 'mzML')
-out <file> Set of all identified peptide groups (i.e. peptide pairs or triplets or singlets or ..).
The m/z-RT positions correspond to the lightest peptide in each group. (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
--help Shows options
--helphelp Shows all options (including advanced)
The following configuration subsections are valid:
- algorithm Parameters for the algorithm.
- labels Isotopic labels that can be specified in section 'sample'.
- sample Parameters describing the sample and its labels.
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">+SILACAnalyzer</span><span class="node_description">Determination of peak ratios in LC-MS data</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 'SILACAnalyzer'</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">Raw LC-MS data to be analyzed. (Profile data required. Will not work with centroided data!)</span><span class="item_tags">input file</span><span class="item_restrictions">*.mzML</span></div> <div class="item"><span class="item_name" style="padding-left:24px;">out</span><span class="item_value"></span>
<span class="item_description">Set of all identified peptide groups (i.e. peptide pairs or triplets or singlets or ..). The m/z-RT positions correspond to the lightest peptide in each group.</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;">out_clusters</span><span class="item_value"></span>
<span class="item_description">Optional debug output containing data points passing all filters, hence belonging to a SILAC pattern. Points of the same colour correspond to the mono-isotopic peak of the lightest peptide in a pattern.</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;">out_features</span><span class="item_value"></span>
<span class="item_description">Optional output file containing the individual peptide features in 'out'.</span><span class="item_tags">output file</span><span class="item_restrictions">*.featureXML</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:24px;">out_mzq</span><span class="item_value"></span>
<span class="item_description">Optional output file of MzQuantML.</span><span class="item_tags">output file</span><span class="item_restrictions">*.mzq</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:24px;">out_filters</span><span class="item_value"></span>
<span class="item_description">Optional output file containing all points that passed the filters as txt. Suitable as input for 'in_filters' to perform clustering without preceding filtering process.</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;">in_filters</span><span class="item_value"></span>
<span class="item_description">Optional input file containing all points that passed the filters as txt. Use output from 'out_filters' to perform clustering only.</span><span class="item_tags">input file</span><span class="item_restrictions">*.consensusXML</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:24px;">out_debug</span><span class="item_value"></span>
<span class="item_description">Filename base for debug output.</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;">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 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">+++algorithm</span><span class="node_description">Parameters for the algorithm.</span></div>
<div class="item item_advanced"><span class="item_name" style="padding-left:32px;">allow_missing_peaks</span><span class="item_value">false</span>
<span class="item_description">Low intensity peaks might be missing from the isotopic pattern of some of the peptides. Should such peptides be included in the analysis?</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;">rt_threshold</span><span class="item_value">30</span>
<span class="item_description">Typical retention time [s] over which a characteristic peptide elutes. (This is not an upper bound. Peptides that elute for longer will be reported.)</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">rt_min</span><span class="item_value">0</span>
<span class="item_description">Lower bound for the retention time [s].</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">intensity_cutoff</span><span class="item_value">1000</span>
<span class="item_description">Lower bound for the intensity of isotopic peaks in a SILAC pattern.</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">intensity_correlation</span><span class="item_value">0.7</span>
<span class="item_description">Lower bound for the Pearson correlation coefficient, which measures how well intensity profiles of different isotopic peaks correlate.</span><span class="item_tags"></span><span class="item_restrictions">0:1</span></div> <div class="item"><span class="item_name" style="padding-left:32px;">model_deviation</span><span class="item_value">3</span>
<span class="item_description">Upper bound on the factor by which the ratios of observed isotopic peaks are allowed to differ from the ratios of the theoretic averagine model, i.e. ( theoretic_ratio / model_deviation ) < observed_ratio < ( theoretic_ratio * model_deviation ).</span><span class="item_tags"></span><span class="item_restrictions">1:∞</span></div> <div class="node"><span class="node_name">+++labels</span><span class="node_description">Isotopic labels that can be specified in section 'sample'.</span></div>
<div class="item item_advanced"><span class="item_name" style="padding-left:32px;">Arg6</span><span class="item_value">6.0201290268</span>
<span class="item_description">Arg6 mass shift</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">Arg10</span><span class="item_value">10.0082686</span>
<span class="item_description">Arg10 mass shift</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">Lys4</span><span class="item_value">4.0251069836</span>
<span class="item_description">Lys4 mass shift</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">Lys6</span><span class="item_value">6.0201290268</span>
<span class="item_description">Lys6 mass shift</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">Lys8</span><span class="item_value">8.0141988132</span>
<span class="item_description">Lys8 mass shift</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">dICPL4</span><span class="item_value">4.025107</span>
<span class="item_description">mass difference between isotope-coded protein labels ICPL 4 and ICPL 0</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">dICPL6</span><span class="item_value">6.020129</span>
<span class="item_description">mass difference between isotope-coded protein labels ICPL 6 and ICPL 0</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">dICPL10</span><span class="item_value">10.045236</span>
<span class="item_description">mass difference between isotope-coded protein labels ICPL 10 and ICPL 0</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">Methyl4</span><span class="item_value">4.0202</span>
<span class="item_description">Methyl4 mass shift</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">Methyl8</span><span class="item_value">8.0202</span>
<span class="item_description">Methyl8 mass shift</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">Methyl12</span><span class="item_value">12.0202</span>
<span class="item_description">Methyl12 mass shift</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">Methyl16</span><span class="item_value">16.0202</span>
<span class="item_description">Methyl16 mass shift</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">Methyl24</span><span class="item_value">24.0202</span>
<span class="item_description">Methyl24 mass shift</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">Methyl32</span><span class="item_value">32.0202</span>
<span class="item_description">Methyl32 mass shift</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="node"><span class="node_name">+++sample</span><span class="node_description">Parameters describing the sample and its labels.</span></div>
<div class="item"><span class="item_name" style="padding-left:32px;">labels</span><span class="item_value">[Lys8,Arg10]</span>
<span class="item_description">Labels used for labelling the sample. [...] specifies the labels for a single sample. For example, [Lys4,Arg6][Lys8,Arg10] describes a mixtures of three samples. One of them unlabelled, one labelled with Lys4 and Arg6 and a third one with Lys8 and Arg10. For permitted labels see 'advanced parameters', section 'labels'. If left empty the tool identifies singlets, i.e. acts as peptide feature finder (in this case, 'out_features' must be used for output instead of 'out').</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">charge</span><span class="item_value">2:4</span>
<span class="item_description">Range of charge states in the sample, i.e. min charge : max charge.</span><span class="item_tags"></span><span class="item_restrictions"> </span></div> <div class="item"><span class="item_name" style="padding-left:32px;">missed_cleavages</span><span class="item_value">0</span>
<span class="item_description">Maximum number of missed cleavages.</span><span class="item_tags"></span><span class="item_restrictions">0:∞</span></div> <div class="item item_advanced"><span class="item_name" style="padding-left:32px;">peaks_per_peptide</span><span class="item_value">3:5</span>
<span class="item_description">Range of peaks per peptide in the sample, i.e. min peaks per peptide : max peaks per peptide. For example 3:6, if isotopic peptide patterns in the sample consist of either three, four, five or six isotopic peaks. </span><span class="item_tags"></span><span class="item_restrictions"> </span></div></div>
<p><b>Parameter Tuning</b></p>
<p>SILACAnalyzer can detect SILAC patterns of any number of peptides, i.e. doublets (pairs), triplets, quadruplets et cetera.</p>
<p><em>input:</em></p>
<ul>
<li>in [*.mzML] - LC-MS dataset to be analyzed</li>
<li>ini [*.ini] - file containing all parameters (see discussion below)</li>
</ul>
<p><em>standard output:</em></p>
<ul>
<li>out [*.consensusXML] - contains the list of identified peptides (retention time and m/z of the lightest peptide, ratios)</li>
</ul>
<p><em>optional output:</em></p>
<ul>
<li>out_clusters [*.consensusXML] - contains the complete set of data points passing the filters, see Fig. (e)</li>
</ul>
<p>The results of an analysis can easily visualized within TOPPView. Simply load *.consensusXML and *.featureXML as layers over the original *.mzML.</p>
<p>Parameters in section <em>algorithm:</em></p>
<ul>
<li><em>allow_missing_peaks</em> - Low intensity peaks might be missing from the isotopic pattern of some of the peptides. Specify if such peptides should be included in the analysis.</li>
<li><em>rt_threshold</em> - Upper bound for the retention time [s] over which a characteristic peptide elutes.</li>
<li><em>rt_min</em> - Lower bound for the retentions time [s].</li>
<li><em>intensity_cutoff</em> - Lower bound for the intensity of isotopic peaks in a SILAC pattern.</li>
<li><em>intensity_correlation</em> - Lower bound for the Pearson correlation coefficient, which measures how well intensity profiles of different isotopic peaks correlate.</li>
<li><em>model_deviation</em> - Upper bound on the factor by which the ratios of observed isotopic peaks are allowed to differ from the ratios of the theoretic averagine model, i.e. ( theoretic_ratio / model_deviation ) < observed_ratio < ( theoretic_ratio * model_deviation ).</li>
</ul>
<p>Parameters in section <em>sample:</em></p>
<ul>
<li><em>labels</em> - Labels used for labelling the sample. [...] specifies the labels for a single sample. For example, [Lys4,Arg6][Lys8,Arg10] describes a mixtures of three samples. One of them unlabelled, one labelled with Lys4 and Arg6 and a third one with Lys8 and Arg10. For permitted labels see section <em>labels</em>.</li>
<li><em>charge</em> - Range of charge states in the sample, i.e. min charge : max charge.</li>
<li><em>missed_cleavages</em> - Maximum number of missed cleavages.</li>
<li><em>peaks_per_peptide</em> - Range of peaks per peptide in the sample, i.e. min peaks per peptide : max peaks per peptide.</li>
</ul>
<p>Parameters in section <em>labels:</em> This section contains a list of all isotopic labels currently available for analysis of SILAC data with SILACAnalyzer.</p>
<p><b>References:</b> <br/>
L. Nilse, M. Sturm, D. Trudgian, M. Salek, P. Sims, K. Carroll, S. Hubbard, <a href="http://www.springerlink.com/content/u40057754100v71t">SILACAnalyzer - a tool for differential quantitation of stable isotope derived data</a>, in F. Masulli, L. Peterson, and R. Tagliaferri (Eds.): CIBB 2009, LNBI 6160, pp. 4555, 2010. </p>
</div></div><!-- contents -->
<HR style="height:1px; border:none; border-top:1px solid #c0c0c0;">
<TABLE width="100%" border="0">
<TR>
<TD><font color="#c0c0c0">OpenMS / TOPP release 1.11.1</font></TD>
<TD align="right"><font color="#c0c0c0">Documentation generated on Thu Nov 14 2013 11:19:24 using doxygen 1.8.5</font></TD>
</TR>
</TABLE>
</BODY>
</HTML>
|
