【1】蛋白質(zhì)組學(xué)鑒定軟件之X!Tandem

1. 簡(jiǎn)介

X!Tandem是GPM：The Global Proteome Machine（主要基于Web的開源用戶界面，用于分析和顯示蛋白質(zhì)鑒定數(shù)據(jù)孤紧。關(guān)于GPM的更多內(nèi)容可參考https://www.thegpm.org/GPM/faq.html）中的一個(gè)成員凌那，此外還有X! P3 唁情、X! Hunter等工具姻僧。顧名思義，X觉痛！表示X! series搜索引擎役衡，Tandem表示串聯(lián)質(zhì)譜，所以X!Tandem用于串聯(lián)質(zhì)譜數(shù)據(jù)分析薪棒。

軟件優(yōu)點(diǎn)：

免費(fèi)開源手蝎，win/linux/mac版本
集群系統(tǒng)可采用Parallel Tandem 或 X!!Tandem并行處理
運(yùn)行速度還可以，c++寫的
使用簡(jiǎn)單：命令行調(diào)用XML文件俐芯，輸出格式（也是XML）適用于GPM其他工具
統(tǒng)計(jì)可靠：除了譜圖匹配肽段序列棵介，它還將數(shù)據(jù)中的所有肽段重新歸并到已知蛋白序列，并且給出統(tǒng)計(jì)證據(jù)（組裝和匹配是非隨機(jī)的）吧史。
不需要PeptideProphet 和 ProteinProphet等額外蛋白歸并和統(tǒng)計(jì)分析軟件邮辽。
蛋白期望值計(jì)算公式：

image.png

官網(wǎng)只提優(yōu)點(diǎn)，沒說缺點(diǎn)贸营。根據(jù)試用的情況吨述，簡(jiǎn)單提幾點(diǎn)：

結(jié)果不友好：還需要后處理
軟件更新慢：最新版ALANINE (2017.02.01)

2.下載安裝

下載地址：
ftp://ftp.thegpm.org/projects/tandem/source
這里只試用Linux版本，下載解壓

tar -xzf tandem-linux-17-02-01-4.zip

解壓后三個(gè)子文件夾：

src包含軟件源碼
bin包含軟件二進(jìn)制文件和示例文件
fasta包含示例蛋白序列文件

image.png

3. 軟件試用

軟件運(yùn)行很簡(jiǎn)單：

/path/bin/tandem.exe  input.xml

input.xml配置文件中調(diào)用了default_input.xml和taxonomy.xml文件钞脂，然后就是物種名揣云，輸入的譜圖文件（需要mgf格式）和輸出文件：

<?xml version="1.0"?>
<bioml>
    <note>
    Each one of the parameters for x! tandem is entered as a labeled note node.
    Any of the entries in the default_input.xml file can be over-ridden by
    adding a corresponding entry to this file. This file represents a minimum
    input file, with only entries for the default settings, the output file
    and the input spectra file name.
    See the taxonomy.xml file for a description of how FASTA sequence list
    files are linked to a taxon name.
    </note>
    <note type="input" label="list path, default parameters">default_input.xml</note>
    <note type="input" label="list path, taxonomy information">taxonomy.xml</note>
    <note type="input" label="protein, taxon">yeast</note>
    <note type="input" label="spectrum, path">test_spectra.mgf</note>
    <note type="input" label="output, path">output.xml</note>
</bioml>

示例中taxonomy.xml文件有多個(gè)物種的序列。而實(shí)際項(xiàng)目中我們往往針對(duì)一個(gè)物種冰啃，因此下載fasta文件配置一個(gè)即可：

<?xml version="1.0"?>
<bioml label="x! taxon-to-file matching list">
    <taxon label="yeast">
        <file format="peptide" URL="../fasta/scd.fasta.pro" />
        <file format="peptide" URL="../fasta/scd_1.fasta.pro" />
        <file format="peptide" URL="../fasta/crap.fasta.pro" />
    </taxon>
</bioml>

串聯(lián)質(zhì)譜的參數(shù)設(shè)置主要在default_input.xml中邓夕，可配置的參數(shù)很多，具體解釋可參考https://www.thegpm.org/TANDEM/api/index.html阎毅。大部分使用默認(rèn)即可焚刚，我們需要設(shè)置往往只有少數(shù)幾個(gè)，比如一二級(jí)誤差及其單位扇调，可變修飾和固定修飾（注意修飾格式的寫法與其他軟件不同矿咕，更多的修飾可查看https://www.unimod.org/login.php?message=expired），漏切數(shù)等等。

<?xml version="1.0"?>
<?xml-stylesheet type="text/xsl" href="tandem-input-style.xsl"?>
<bioml>
<note>list path parameters</note>
    <note type="input" label="list path, default parameters">default_input.xml</note>
        <note>This value is ignored when it is present in the default parameter
        list path.</note>
    <note type="input" label="list path, taxonomy information">taxonomy.xml</note>

<note>spectrum parameters</note>
    <note type="input" label="spectrum, fragment monoisotopic mass error">0.4</note>
    <note type="input" label="spectrum, parent monoisotopic mass error plus">100</note>
    <note type="input" label="spectrum, parent monoisotopic mass error minus">100</note>
    <note type="input" label="spectrum, parent monoisotopic mass isotope error">yes</note>
    <note type="input" label="spectrum, fragment monoisotopic mass error units">Daltons</note>
    <note>The value for this parameter may be 'Daltons' or 'ppm': all other values are ignored</note>
    <note type="input" label="spectrum, parent monoisotopic mass error units">ppm</note>
        <note>The value for this parameter may be 'Daltons' or 'ppm': all other values are ignored</note>
    <note type="input" label="spectrum, fragment mass type">monoisotopic</note>
        <note>values are monoisotopic|average </note>

<note>spectrum conditioning parameters</note>
    <note type="input" label="spectrum, dynamic range">100.0</note>
        <note>The peaks read in are normalized so that the most intense peak
        is set to the dynamic range value. All peaks with values of less that
        1, using this normalization, are not used. This normalization has the
        overall effect of setting a threshold value for peak intensities.</note>
    <note type="input" label="spectrum, total peaks">50</note>
        <note>If this value is 0, it is ignored. If it is greater than zero (lets say 50),
        then the number of peaks in the spectrum with be limited to the 50 most intense
        peaks in the spectrum. X! tandem does not do any peak finding: it only
        limits the peaks used by this parameter, and the dynamic range parameter.</note>
    <note type="input" label="spectrum, maximum parent charge">4</note>
    <note type="input" label="spectrum, use noise suppression">yes</note>
    <note type="input" label="spectrum, minimum parent m+h">500.0</note>
    <note type="input" label="spectrum, minimum fragment mz">150.0</note>
    <note type="input" label="spectrum, minimum peaks">15</note>
    <note type="input" label="spectrum, threads">1</note>
    <note type="input" label="spectrum, sequence batch size">1000</note>

<note>residue modification parameters</note>
    <note type="input" label="residue, modification mass">57.022@C</note>
        <note>The format of this parameter is m@X, where m is the modfication
        mass in Daltons and X is the appropriate residue to modify. Lists of
        modifications are separated by commas. For example, to modify M and C
        with the addition of 16.0 Daltons, the parameter line would be
        +16.0@M,+16.0@C
        Positive and negative values are allowed.
        </note>
    <note type="input" label="residue, potential modification mass"></note>
        <note>The format of this parameter is the same as the format
        for residue, modification mass (see above).</note>
    <note type="input" label="residue, potential modification motif"></note>
        <note>The format of this parameter is similar to residue, modification mass,
        with the addition of a modified PROSITE notation sequence motif specification.
        For example, a value of 80@[ST!]PX[KR] indicates a modification
        of either S or T when followed by P, and residue and the a K or an R.
        A value of 204@N!{P}[ST]{P} indicates a modification of N by 204, if it
        is NOT followed by a P, then either an S or a T, NOT followed by a P.
        Positive and negative values are allowed.
        </note>

<note>protein parameters</note>
    <note type="input" label="protein, taxon">other mammals</note>
        <note>This value is interpreted using the information in taxonomy.xml.</note>
    <note type="input" label="protein, cleavage site">[RK]|{P}</note>
        <note>this setting corresponds to the enzyme trypsin. The first characters
        in brackets represent residues N-terminal to the bond - the '|' pipe -
        and the second set of characters represent residues C-terminal to the
        bond. The characters must be in square brackets (denoting that only
        these residues are allowed for a cleavage) or french brackets (denoting
        that these residues cannot be in that position). Use UPPERCASE characters.
        To denote cleavage at any residue, use [X]|[X] and reset the
        scoring, maximum missed cleavage site parameter (see below) to something like 50.
        </note>
    <note type="input" label="protein, modified residue mass file"></note>
    <note type="input" label="protein, cleavage C-terminal mass change">+17.002735</note>
    <note type="input" label="protein, cleavage N-terminal mass change">+1.007825</note>
    <note type="input" label="protein, N-terminal residue modification mass">0.0</note>
    <note type="input" label="protein, C-terminal residue modification mass">0.0</note>
    <note type="input" label="protein, homolog management">no</note>
        <note>if yes, an upper limit is set on the number of homologues kept for a particular spectrum</note>

<note>model refinement parameters</note>
    <note type="input" label="refine">yes</note>
    <note type="input" label="refine, modification mass"></note>
    <note type="input" label="refine, sequence path"></note>
    <note type="input" label="refine, tic percent">20</note>
    <note type="input" label="refine, spectrum synthesis">yes</note>
    <note type="input" label="refine, maximum valid expectation value">0.1</note>
    <note type="input" label="refine, potential N-terminus modifications"></note>
    <note type="input" label="refine, potential C-terminus modifications"></note>
    <note type="input" label="refine, unanticipated cleavage">yes</note>
    <note type="input" label="refine, potential modification mass"></note>
    <note type="input" label="refine, point mutations">no</note>
    <note type="input" label="refine, use potential modifications for full refinement">no</note>
    <note type="input" label="refine, point mutations">no</note>
    <note type="input" label="refine, potential modification motif"></note>
    <note>The format of this parameter is similar to residue, modification mass,
        with the addition of a modified PROSITE notation sequence motif specification.
        For example, a value of 80@[ST!]PX[KR] indicates a modification
        of either S or T when followed by P, and residue and the a K or an R.
        A value of 204@N!{P}[ST]{P} indicates a modification of N by 204, if it
        is NOT followed by a P, then either an S or a T, NOT followed by a P.
        Positive and negative values are allowed.
        </note>

<note>scoring parameters</note>
    <note type="input" label="scoring, minimum ion count">4</note>
    <note type="input" label="scoring, maximum missed cleavage sites">1</note>
    <note type="input" label="scoring, x ions">no</note>
    <note type="input" label="scoring, y ions">yes</note>
    <note type="input" label="scoring, z ions">no</note>
    <note type="input" label="scoring, a ions">no</note>
    <note type="input" label="scoring, b ions">yes</note>
    <note type="input" label="scoring, c ions">no</note>
    <note type="input" label="scoring, cyclic permutation">no</note>
        <note>if yes, cyclic peptide sequence permutation is used to pad the scoring histograms</note>
    <note type="input" label="scoring, include reverse">no</note>
        <note>if yes, then reversed sequences are searched at the same time as forward sequences</note>
    <note type="input" label="scoring, cyclic permutation">no</note>
    <note type="input" label="scoring, include reverse">no</note>

<note>output parameters</note>
    <note type="input" label="output, log path"></note>
    <note type="input" label="output, message">testing 1 2 3</note>
    <note type="input" label="output, one sequence copy">no</note>
    <note type="input" label="output, sequence path"></note>
   <note type="input" label="output, path">output.xml</note>
    <note type="input" label="output, sort results by">protein</note>
        <note>values = protein|spectrum (spectrum is the default)</note>
    <note type="input" label="output, path hashing">yes</note>
        <note>values = yes|no</note>
    <note type="input" label="output, xsl path">tandem-style.xsl</note>
    <note type="input" label="output, parameters">yes</note>
        <note>values = yes|no</note>
    <note type="input" label="output, performance">yes</note>
        <note>values = yes|no</note>
    <note type="input" label="output, spectra">yes</note>
        <note>values = yes|no</note>
    <note type="input" label="output, histograms">yes</note>
        <note>values = yes|no</note>
    <note type="input" label="output, proteins">yes</note>
        <note>values = yes|no</note>
    <note type="input" label="output, sequences">yes</note>
        <note>values = yes|no</note>
    <note type="input" label="output, one sequence copy">no</note>
        <note>values = yes|no, set to yes to produce only one copy of each protein sequence in the output xml</note>
    <note type="input" label="output, results">valid</note>
        <note>values = all|valid|stochastic</note>
    <note type="input" label="output, maximum valid expectation value">0.1</note>
        <note>value is used in the valid|stochastic setting of output, results</note>
    <note type="input" label="output, histogram column width">30</note>
        <note>values any integer greater than 0. Setting this to '1' makes cutting and pasting histograms
        into spread sheet programs easier.</note>
<note type="description">ADDITIONAL EXPLANATIONS</note>
    <note type="description">Each one of the parameters for X! tandem is entered as a labeled note
            node. In the current version of X!, keep those note nodes
            on a single line.
    </note>
    <note type="description">The presence of the type 'input' is necessary if a note is to be considered
            an input parameter.
    </note>
    <note type="description">Any of the parameters that are paths to files may require alteration for a
            particular installation. Full path names usually cause the least trouble,
            but there is no reason not to use relative path names, if that is the
            most convenient.
    </note>
    <note type="description">Any parameter values set in the 'list path, default parameters' file are
            reset by entries in the normal input file, if they are present. Otherwise,
            the default set is used.
    </note>
    <note type="description">The 'list path, taxonomy information' file must exist.
        </note>
    <note type="description">The directory containing the 'output, path' file must exist: it will not be created.
        </note>
    <note type="description">The 'output, xsl path' is optional: it is only of use if a good XSLT style sheet exists.
        </note>

</bioml>

4. 結(jié)果

軟件得到的結(jié)果也是xml文件痴腌，文件名會(huì)自動(dòng)加上運(yùn)行的日期雌团，如output.2020_07_03_15_56_03.t.xml。對(duì)格式需要有一定了解才能從中有效的提取信息士聪，關(guān)于輸出的文件格式解釋，參考這份文檔：https://www.thegpm.org/docs/X_series_output_form.pdf

這里從我跑過的一個(gè)項(xiàng)目中截取一小部分結(jié)果來做說明：

<?xml version="1.0"?>
<?xml-stylesheet type="text/xsl" href="tandem-style.xsl"?>
<bioml xmlns:GAML="http://www.bioml.com/gaml/" label="models from '/path/test.mgf'">
<group id="373508" mh="1842.861248" z="3" rt="6989.4672" expect="8.7e-12" label="[denovogenes]_384740" type="model" sumI="7.12" maxI="1.08216e+06" fI="10821.6" act="0" >
<protein expect="-196.9" id="373508.1" uid="2902844" label="[denovogenes]_384740" sumI="9.09" >
<note label="description">[denovogenes]_384740</note>
<file type="peptide" URL="/path/test.fa"/>
<peptide start="1" end="434">
    MSIITDVYAR EVLDSRGNPT LEVEVYTESG AFGRGMVPSG ASTGEHEAVE
    LRDGDKARYG GLGTQKAVDN VNNVIAEHII GFDVRDQQGI DRAMIALDGT
    PNKGKLGANA ILGVSIAVAR AAADYLEVPL YSYLGGFNTK VLPTPMMNII
    NGGSHSDAPI AFQEFMIVPA GAPTFKEALR WGAEIFHALK KILKERGLET
    AVGDEGGFAP RFDGTEDGVE TIIKAIEAAG YVPGKDVFIG FDCASSEFYD
    AERKVYDYTK FEGEGAAVRT AAEQIDYLEE LVNKYPIITI EDGMDENDWD
    GWKALTERLG GKVQLVGDDF FVTNTAYLEK GIAEHAANSI LIKVNQIGTL
    TETFDAIEMA KEAGYTAVVS HRSGETEDST IADIAVATNA GQIKTGSLSR
    TDRIAKYIQL LRIEEQLGEV AEYRGLKSFY NLKK
<domain id="373508.1.1" start="35" end="52" expect="8.7e-12" mh="1842.8650" delta="-0.0038" hyperscore="83.7" nextscore="45.9" y_score="9.5" y_ions="23" b_score="10.1" b_ions="6" pre="AFGR" post="DGDK" seq="GMVPSGASTGEHEAVELR" missed_cleavages="0">
<aa type="M" at="36" modified="15.99492" />
</domain>
</peptide>
......

假設(shè)我只提取蛋白ID猛蔽，delta剥悟，domain起始和終止，期望和序列等信息曼库。xml格式在各種編程語言中都有解析包区岗，自己寫腳本提取后可得到如下形式，再進(jìn)行后續(xù)處理（如宏蛋白中優(yōu)化數(shù)據(jù)庫(kù)毁枯，將鑒定的蛋白ID匹配回原始數(shù)據(jù)庫(kù)慈缔，從而達(dá)到優(yōu)化數(shù)據(jù)庫(kù)的目的）。

image.png

5. FAQ

最常見的FAQ官網(wǎng)已經(jīng)給出种玛，基本上這些也夠了藐鹤。
https://www.thegpm.org/TANDEM/tandem_install_faq.html
主要是理解三個(gè)輸入的配置文件input.xml，taxonomy.xml赂韵，default_input.xml以及知道它們干什么的娱节。

蛋白質(zhì)組學(xué)鑒定定量軟件總結(jié)：
【1】蛋白質(zhì)組學(xué)鑒定軟件之X!Tandem
【2】蛋白質(zhì)組學(xué)鑒定軟件之Comet
【3】蛋白質(zhì)組學(xué)鑒定軟件之Mascot
【4】蛋白質(zhì)組學(xué)鑒定軟件之MSGFPlus
【5】蛋白質(zhì)組學(xué)鑒定定量軟件之PD
【6】蛋白質(zhì)組學(xué)鑒定定量軟件之MaxQuant

最后編輯于：2020.09.01 09:30:33

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