【1】蛋白鉴定软件之X!Tandem

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所属分类:生物信息学

目录
1. 简介
X!Tandem是(主要基于Web的开源用户界面,用于分析和显示蛋白质鉴定数据。关于GPM的更多内容可参考)中的一个成员,此外还有X! P3 、X! Hunter等工具。顾名思义,X!表示X! series搜索引擎,Tandem表示串联质谱,所以X!Tandem用于串联质谱数据分析。
软件优点:
免费开源,win/linux/mac版本
集群系统可采用 或 并行处理
运行速度还可以,c++写的
使用简单:命令行调用XML文件,输出格式(也是XML)适用于GPM其他工具
统计可靠:除了谱图匹配肽段序列,它还将数据中的所有肽段重新归并到已知蛋白序列,并且给出统计证据(组装和匹配是非随机的)。
不需要PeptideProphet 和 ProteinProphet等额外蛋白归并和统计分析软件。
蛋白期望值计算公式:

官网只提优点,没说缺点。根据试用的情况,简单提几点:
结果不友好:还需要后处理
软件更新慢:最新版ALANINE (2017.02.01)
2.下载安装
下载地址:

这里只试用Linux版本,下载解压
tar -xzf tandem-linux-17-02-01-4.zip

解压后三个子文件夹:
src包含软件源码
bin包含软件二进制文件和示例文件
fasta包含示例蛋白序列文件

3. 软件试用
软件运行很简单:
/path/bin/tandem.exe input.xml

input.xml配置文件中调用了default_input.xml和taxonomy.xml文件,然后就是物种名,输入的谱图文件(需要mgf格式)和输出文件:
?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文件有多个物种的序列。而实际项目中我们往往针对一个物种,因此下载fasta文件配置一个即可:
?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

串联质谱的参数设置主要在default_input.xml中,可配置的参数很多,具体解释可参考。大部分使用默认即可,我们需要设置往往只有少数几个,比如一二级误差及其单位,可变修饰和固定修饰(注意修饰格式的写法与其他软件不同,更多的修饰可查看),漏切数等等。
?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. 结果
软件得到的结果也是xml文件,文件名会自动加上运行的日期,如output.2020_07_03_15_56_03.t.xml。对格式需要有一定了解才能从中有效的提取信息,关于输出的文件格式解释,参考这份文档:
这里从我跑过的一个项目中截取一小部分结果来做说明:
?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
......

假设我只提取蛋白ID,delta,domain起始和终止,期望和序列等信息。xml格式在各种编程语言中都有解析包,自己写脚本提取后可得到如下形式,再进行后续处理(如宏蛋白中优化数据库,将鉴定的蛋白ID匹配回原始数据库,从而达到优化数据库的目的)。

5. FAQ
最常见的FAQ官网已经给出,基本上这些也够了。

主要是理解三个输入的配置文件input.xml,taxonomy.xml,default_input.xml以及知道它们干什么的。
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