TPP:Developer Documentation

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Contents

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License

All of the TPP is released under the LGPL license. Some other programs (converters) have different licences.


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Languages

C, C++, perl


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Version control

This project is hosted at sourceforge ([1]) under the "sashimi" project, using SVN.


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SVN access

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Anonymous access

Anyone can check out the code: ...

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Developer access

You'll need to be a project developer: register at SourceForge and contact one of the main Sashimi developers. 

 see our SourceForge SVN info page.
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XML Parsing libraries

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perl

  • xml tree
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c++

  • A. Keller's "tag" system:

All Parser programs (children of Parser.cxx) parse pepXML and protXML with the constrain that all text be enclosed within a 'tag' such as the following example: 

<mytag name="akeller"><email address="akeller@systemsbiology.org/></mytag>

NOT ACCEPTABLE are files with text outside of a bracket tag enclosure, such as 'akeller' in the following illegal example: 

<illegaltag>akeller</illegaltag>
  • J. Tasman code


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System Requirements

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Webserver

Webserver with access to data directories. See configuration section below. Apache support is the default configuration and is installed and configured on port 1441 automatically as part of the current cygwin installation. IIS on windows is no longer a supported configuration.

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XSLT Processor

The TPP currently relies on an "xsl transform" processor for manipulating XML data.

  1. One such program, xsltproc, is usually distributed with Linux so you most likely already have it. It should reside in the /usr/bin/ directory. If it is not already on your computer, first try to install it via the standard package system for your distribution, or the cygwin installer for cygwin. Otherwise you can download it for free at: http://xmlsoft.org/XSLT/downloads.html
  2. Another freely available XSLT processor, Xalan, will also work fine with ProteinProphet. If you use it, just make sure it is installed in a directory already on the library path, or else set the LD_LIBRARY_PATH variable to include its location on the webserver: 
Add LD_LIBRARY_PATH files to /etc/ld.so.conf
Then type: ldconfig -v

The viewing of large xml files is sometimes slow. We are hoping to optimize the stylesheets in the future, and in some cases, bypass XSLT altogether.

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Libraries

  • expat-2.0.0 (copied to svn tree)
  • boost 1.32
  • xerces (version?)

You will need to install the following C libraries. These libraries are very common on linux distributions. Make sure that you do not already have them before trying to install. If you do need to install, first try to use the standard package system (e.g. RPMs for Fedora linux , cygwin installer for cygwin-- see below, etc.) If you cannot install them via the normal package system for your distribution, go to their website and download directly.

  • libgd www.boutell.com/gd
  • libpng www.libpng.org
  • zlib www.gzip.org/zlib

Windows users should get this by using the Cygwin installer (www.cygwin.com). Make sure to get the devel packages, in order to have the required .h files.

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Building from source

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Configuration

On TPP:Documentation you can see other guides for building on specific systems.

Skip ahead to Compilation for a Cygwin build, the following is for Linux only. Note that we distribute precompiled binaries in a complete custom cygwin installation.

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Edit the src/Makefile.incl file

Set the TPP_ROOT variable to the directory where you want to install the TPP. Include the trailing '/' when setting the path, e.g.: 

TPP_ROOT=/usr/local/tpp/

Set the TPP_WEB variable to the webserver root relative alias to the TPP. Include the trailing '/' when setting the path, e.g.: 

TPP_WEB=/tpp/

WARNING: To avoid problems during the installation 'you MUST include the trailing '/' when setting the above two paths'.

  1. Set the XSLT_PROC to the path of the xsltproc executable on your system 
XSLT_PROC=/usr/bin/xsltproc

In the src directory, type 'make configure' (once again, DON'T DO THIS for Windows/Cygwin!!!)

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Compilation

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Linux

In the src directory: type 'make all' to compile binaries

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Windows/Cygwin

In the src directory: type 'make windows' to compile binaries
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Installation

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Linux

In the src directory: type 'make install' to install all the binaries

The TPP will be installed in the following directory structure by default:

/usr/local/tpp
TPP root directory
/usr/local/tpp/cgi-bin
CGI-BIN for tpp, contains all web served executables
/usr/local/tpp/bin 
binary directory
/usr/local/tpp/html 
contains all non-executable web served objects
/usr/local/tpp/etc 
contains miscelaneous configuration files
/usr/local/tpp/schema
contains all XML schema files
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Windows/Cygwin

In the src directory: type 'make install-windows' to install all the binaries



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Webserver Configuration

Ideally, all data directories should be cross mounted under the webserver root. Webserver should have SSI (server side includes) turned on. If it is not already, you can activate the Web Server SSI (Server Side Includes)

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Activating SSI on Linux

Modify the /etc/httpd.conf file:

In the document root <Directory> section, add +Includes to the end of already existing Options line: 

 Options +Includes

Uncomment or add in the mod_mime.c section: 

AddType text/html .shtml
AddHandler server-parsed  .shtml

Then restart web server: /etc/rc.d/init.d/httpd restart


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Webserver Root

The environment variable WEBSERVER_ROOT must be set for the program user(s) as well as the webserver. The WEBSERVER_ROOT should point to the webserver's document root directory (e.g. /home/httpd/html on linux/apache, or ??? on windows/IIS ).

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Apache specific webserver configuration

Configure the webserver:

Add the appropriate web paths to the TPP as described below. If you are using the Apache http server, edit the active 'httpd.conf' file. Add the following Alias and ScriptAlias Directives as described below. Be sure to link to the appropriate tpp-version number. 

#
# ISB-Tools Trans Proteomic Pipeline directives
#


Alias /tpp/html "/usr/local/tpp/html"


<Directory "/usr/local/tpp/html">
   AllowOverride None
   Options Includes Indexes FollowSymLinks MultiViews
   Order allow,deny
   Allow from all
</Directory>


<Directory "/usr/local/tpp/schema">
   AllowOverride None
   Options Includes Indexes FollowSymLinks MultiViews
   Order allow,deny
   Allow from all
</Directory>


ScriptAlias /tpp/cgi-bin/ "/usr/local/tpp/cgi-bin/"


<Directory "/usr/local/tpp/cgi-bin">
   AllowOverride AuthConfig Limit
   Options ExecCGI
   Order allow,deny
   Allow from all
   SetEnv WEBSERVER_ROOT /home/httpd/html
</Directory>
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Windows/Cygwin Apache-specific configuration

Configure as described in "Post-Install Configuration" on the following page: [2]

Apache is installed and configured automatically as part of the Cygwin install.

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XML File Validation

The SAX Validator will use the schema location indicated in the XML file to validate: 

SAX2Count -v=always myfile.xml


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Running the TPP

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Overview

Specific instructions are geared towards command-line usage. Note that the web-base GUI is a very convenient way to do these steps.

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Conversion of raw spectroscopy data to mzXML format

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Assigning peptide sequences to spectra (using a search engine)

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Converting search engine results to pepXML format

Analysis programs begin with a pepXML-format file called 'summary.xml', containing the peptides identified by the search engine.


  • For SEQUEST results, you must specify the sequest.params file used for the search:

In directory with summary.html and summary.mzXML (as well as the SEQUEST results .tgz or subdirectory), type: 

Sequest2XML summary.html -Psequest.params
  • For Mascot results, you must specify the database used for search:

In the directory with summary.dat and summary.mzXML, type: 

Mascot2XML summary.dat -D/full/path/database

You can view the search results by opening the 'summary.xml' file in your browser.

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Processing peptide data with the pipeline (using xinteract)

Next, you can run xinteract to apply all or some parts of the pipeline. Type 'xinteract' with no arguments for usage instructions. You can also convert and run the pipeline in one step. See xinterct instructions for details.


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Example

To run the pipeline manually, starting with file1.xml and file2.xml:

  1. Combine together data from 2 files:
  2. InteractParser interact.xml file1.xml file2.xml

Peptide Results can be viewed at any point along the analysis by opening the interact.shtml link.

  1. Run PeptideProphet 
PeptideProphetParser interact.xml
  1. Run XPRESS 
XPressPeptideParser interact.xml
  1. Run ASAPRatio 
ASAPRatioPeptideParser interact.xml
  1. Go into database to retrieve all proteins corresponding to identified peptides 
RefreshParser interact.xml /full/path/database
  1. ProteinProphet 
ProteinProphet.pl interact.xml interact-prot.shtml XML_INPUT

From this point on, all analysis is on the output from ProteinProphet: interact-prot.xml Protein Results can be viewed at any point along the analysis by opening the interact-prot.shtml link.

  1. XPRESS Protein 
XPressProteinParser interact-prot.xml
  1. ASAPRatio Protein 
ASAPRatioProteinRatioParser interact-prot.xml
  1. ASAPRatio Pvalue 
ASAPRatioPvalueParser interact-prot.xml

Questions? Search the newsgroup first, then post questions.

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Converters to mzXML

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Converters to pepXML

All converted pepXML files reference a standard sytlesheed pepXML_std.xsl to enable a view of the xml file directly in a browser.

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Sequest2XML

retired in favor of Out2XML 

Sequest2XML summary.html (-P/full/path/mysequest.params) (-M) (-m) (-a) (-pI) (-Eenzyme)

Converts summary.html to summary.xml in pepXML format. Uses sequest.params file in current directory, unless specified as second argument

OPTIONS
-M 
MALDI mode: do not include spectrum spot number in mzXML file name
-m 
monoisotopic masses (regardless of sequest.params setting)
-a 
average masses (regardless of sequest.params setting)
-pI 
compute peptide pI values
-Eenzyme 
set sample enzyme (default is trypsin, possible values are: nonspecific, chymotrypsin, elastase, gluc, gluc_bicarb, aspn, tca, cnbr, trypsin/cnbr, clostripain, iodosobenzoate, protein_endopeptidase, staph_protease, trypsin_k, trypsin_r)
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Mascot2XML

Mascot2XML summary.dat -D/full/path/mydatabase.fasta (-pI) (-Eenzyme)

Converts summary.dat to summary.xml in pepXML format.

OPTIONS
See Sequest2XML for option definitions
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Comet2XML

Comet2XML summary.cmt.tar.gz (-Eenzyme)
OPTIONS
See Sequest2XML for option definitions
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Peptide-level analyses

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InteractParser

InteractParser interact.xml file1.xml file2.xml file3.xml .....

Merges together pepXML files file1.xml, file2.xml, file3.xml .... into interact.xml. Combines all analysis_summary elements, and reindexes spectrum_query elements. Makes a system call to pepxml2html.pl (pepxml2html.pl -file interact.xml) to create stylesheet for viewing interact.xml amd interact.shtml in a browser.

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DatabaseParser

DatabaseParser interact.xml

Prints the database(s) referenced in pepXML document

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RefreshParser

RefreshParser interact.xml /full/path/database.fasta

Goes into database to find all proteins corresponding to identified peptides and overwrites results to interact.xml

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EnzymeDigestionParser

EnzymeDigestionParser interact.xml (-Eenzyme)

Computes number of tolerable termini and number of missed cleavages in dataset using sample enzyme stored in interact.xml unless specified as argument.

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PeptideProphetParser

PeptideProphetParser interact.xml (EXCLUDE) (LEAVE) (ICAT) (NOICAT) (ZERO) (GLYC) (MALDI) (MINPROB=xx)

Runs PeptideProphet with options and overwrites results to interact.xml

OPTIONS
EXCLUDE
exclude delta stars (SEQUEST)
LEAVE
leave delta star values alone (SEQUEST)
ICAT
use peptide icat info in probability calculation
NOICAT
do not use peptide icat info in probability calculation
ZERO
do not discard any data
GLYC
use peptide NXS/T motif info in probability calculation
MALDI
specify maldi spectra
PI
use pI information
ACCMASS
use accurate mass binning
MINPROB=xx
filter away results with a probability less than xx
EXTRAITRS=xx
specify additional EM iterations
NONTT
Do not use NTT information
CLEVEL=xx
Specify Conservative Level
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XPressPeptideParser

XPressPeptideParser interact.xml (-b) (-n<str>,<num>) (-n<str>,<num>) (-n<str>,<num>) (-L or -H)

Runs XPRESS with options and overwrites results to interact.xml

OPTIONS
-m<num> 
change XPRESS mass tolerance (default=1.0)
-l<str> 
change labeled residues (default='C')
-r<num> 
change XPRESS residue mass difference (default=9.0)
-n<str>,<num> 
when specifying multiple isotopic labels, use this option e.g. -nK,3.0 -nL,3.0
-r<num> 
change XPRESS residue mass difference (default=9.0)
-b 
heavy labeled peptide elutes before light labeled partner
-L 
for ratio, set/fix light to 1, vary heavy
-H 
for ratio, set/fix heavy to 1, vary light
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ASAPRatioPeptideParser

ASAPRatioPeptideParser interact.xml (-b) (-l<str>) (-S) (-m<str>) (-F) (-C)

Runs ASAPRatio with options and overwrites results to interact.xml

OPTIONS
-l<str> 
change labeled residues (default='C')
-b 
heavy labeled peptide elutes before light labeled partner
-f<num> 
areaFlag set to num (ratio display option)
-S 
static modification quantification (i.e. each run is either all light or all heavy)
-F 
use fixed scan range for light and heavy
-C 
quantitate only the charge state where the CID was made
-m<str> 
specified label masses (e.g. M74.325Y125.864), only relevant for static modification quantification
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LibraPeptideParser

LibraPeptideParser interact.xml -clibra_condition.xml

Runs LIBRA using channel information specified in libra_condition.xml file and overwrites results to interact.xml

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CompactParser

CompactParser file.xml

Compacts either pepXML or protXML, combining together start and end tags when no elements are contained between them

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Protein-level analyses

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ProteinProphet.pl

ProteinProphet.pl '<interact pep prob html file1><interact pep prob html file2>....' <outfile> (ICAT) (GLYC) (XPRESS) (ASAP_PROPHET) (ACCURACY) (ASAP) (REFRESH) (DELUDE) (NOOCCAM)
OPTIONS
NOOCCAM
non-conservative maximum protein list
ICAT
highlight peptide cysteines
GLYC
highlight peptide N-glycosylation motif
ACCURACY
min pep prob 0
ASAP
compute ASAP ratios for protein entries (ASAP must have been run previously on interact dataset)
REFRESH
import manual changes to ASAP ratios (after initially using ASAP option)
ASAP_PROPHET
*New and Improved* compute ASAP ratios for protein entries (ASAP must have been run previously on all input interact datasets with mz/XML raw data format)
DELUDE
do NOT use peptide degeneracy information when assessing proteins
HTML
write output to static html page (rather than dynamic shtml)
Other options in conjunction with HTML
EXCELPEPS
write output tab delim xls file including all peptides
EXCELxx
write output tab delim xls file including all protein (group)s with minimum probability xx, where xx is a number between 0 and 1
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XPressProteinRatioParser

XPressProteinRatioParser interact-prot.xml

Runs XPRESS and overwrites results to interact-prot.xml

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ASAPRatioProteinRatioParser

ASAPRatioProteinRatioParser interact-prot.xml

Runs ASAPRatio and overwrites results to interact-prot.xml

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LibraProteinRatioParser

interact-prot.xml <normalization_channel>

Runs LIBRA using normalizing ratios to normalization_channel and overwrites results to interact-prot.xml

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Wrappers

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xinteract

xinteract (generaloptions) (-Oprophetoptions) (-Xxpressoptions) (-Aasapoptions) (-L<conditionfile>libraoptions) xmlfile1 xmlfile2 ....
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options
generaloptions
-Nmyfile.xml 
write output to file 'myfile.xml'
-nI 
do not run Interact (convert to pepXML only)
-nP 
do not run PeptideProphet
-nR 
do not run get all proteins corresponding to degenerate peptides from database
-p0 
do not discard search results with PeptideProphet probabilities below 0.05
-x<num> 
number of extra PeptideProphet interations; default <num>=0
-p<num> 
filter results below PeptideProphet probability <num>; default <num>=0.05
-mw 
calculate protein molecular weights
-MONO 
calculate monoisotopic peptide masses during conversion to pepXML
-AVE 
calculate average peptide masses during conversion to pepXML
-eX 
specify sample enzyme other than trypsin
-eC 
specify sample enzyme = Chymotrypsin
-eA 
specify sample enzyme = AspN
-eG 
specify sample enzyme = GluC
-eB 
specify sample enzyme = GluC Bicarb
-eM 
specify sample enzyme = CNBr
-eD 
specify sample enzyme = Trypsin/CNBr
-e3 
specify sample enzyme = Chymotrypsin/AspN/Trypsin
-eE 
specify sample enzyme = Elastase
-eL 
specify sample enzyme = LysN (cuts before K)
-eP 
specify sample enzyme = LysN Promisc (cuts before KASR)
-eN 
specify sample enzyme = Nonspecific or None

For developers:

-t 
run regression test against a previously derived result
-t! 
learn results for regression test
prophetoptions (following the 'O')
i 
use icat information in PeptideProphet
f 
do not use icat information in PeptideProphet
g 
use N-glyc motif information in PeptideProphet
m 
maldi data
I 
use pI information in PeptideProphet
A 
use accurate mass binning in PeptideProphet
w 
warning instead of exit with error if instrument types between runs is different
x 
exclude all entries with asterisked score values in PeptideProphet
l 
leave alone all entries with asterisked score values in PeptideProphet
p 
run ProteinProphet afterwards
u 
do not assemble protein groups in ProteinProphet analysis
s 
do not use Occam's Razor in ProteinProphet analysis to derive the simplest protein list to explain observed peptides
xpressoptions (will run XPRESS analysis with any specified options that follow the 'X')
-m<num> 
change XPRESS mass tolerance (default=1.0)
-l<str> 
change labeled residues (default='C')
-n<str>,<num> 
change XPRESS residue mass difference for <str> to <num> (default=9.0)
-b 
heavy labeled peptide elutes before light labeled partner
-L 
for ratio, set/fix light to 1, vary heavy
-H 
for ratio, set/fix heavy to 1, vary light
asapoptions (will run ASAPRatio analysis with any specified options that follow the 'A')
-l<str>  
change labeled residues (default='C')
-b  
heavy labeled peptide elutes before light labeled partner
-f<num>  
areaFlag set to num (ratio display option)
-S  
static modification quantification (i.e. each run is either all light or all heavy)
-F  
use fixed scan range for light and heavy
-C  
quantitate only the charge state where the CID was made
-m<str>  
specified label masses (e.g. M74.325Y125.864), only relevant for static modification quantification
libraoptions (will run Libra Quantitation analysis with any specified options that follow the 'L')
-<num>  
normalization channel (for protein level quantitation)
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examples
xinteract *.xml
combines together data in all pepXML files into 'interact.xml', then runs PeptideProphet]
xinteract -Ndata.xml *.xml
same as above, but results are written to 'data.xml'
xinteract -Ndata.xml -X -Op *.xml
same as above, but run XPRESS analysis in its default mode, then ProteinProphet
xinteract -X -A file1.xml file2.xml
combines together data in file1.xml and file2.xml into 'interact.xml' and then runs XPRESS (in its default mode) and ASAPRatio (in its default mode)
xinteract -X-nC,6.0 -A file1.xml file2.xml
same as above, but specifies that cysteine label has a heavy/light mass difference of 6.0
xinteract -X -A-lDE-S file1.xml file2.xml
same as above, but specifies for ASAP to run in static mode with labeled residues D and E
xinteract -Lmyconditionfile.xml-1 -Op file1.xml file2.xml
run libra quantitiation after PeptideProphet using myconditionfile.xml, and after ProteinProphet normalizing ratios to channel 1 values
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runpropet

Runs ProteinProphet on designated 'interact.xml' file(s), where interact.xml is a pepXML-formated output file from PeptideProphet.

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usage 1: specify input file and options
runprophet -Ooptions <interact file (with probs)>

runs analysis on inputfile with specified options and writes analysis to inputfile-prot.htm

options
i
icat data (color Cysteines)
g
N-glycosylation data (color NXS/T)
m
multifiles (more than one interact.xml file, must specify outfile
d
delude (do not look up ALL prots corresponding to degenerate peps)
l
use html input files (pre-TPP)
X
import XPRESS protein ratios
A
import ASAPRatio protein ratios and pvalues
L<num>
import Libra protein ratios normalized to channel <num>
a
import ASAPRatio results present in file (starting from scratch)
r
update changes made to ASAPRatio results (previously run using 'a' option)
n
don't use occam's razor for degenerate peps (get max prot list, including many false positives)
u
do not assemble PROTEIN GROUPS
z
do not include zero probability protein entries in output
H
writes results to static html file (and tab delimited excel file)
P
includes peptides in tab delimited excel file (must accompany 'H')
xx
includes results in tab delimited excel file with minimum probability xx, where xx is a number between 0 and 1 (must accompany 'H')
examples
runprophet -OiXA interact.xml
for icat data with mzXML XPRESS and ASAPRatio quantitation information
runprophet -Oia interact.xml
for icat data with non-mzXML ASAPRAtio information
runprophet -Oi interact.xml
for icat data
runprophet -Og interact.xml
for N-glycosylated data
runprophet -OL interact.htm
for pre-TPP html input file
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usage 2: specify input file and use default options
runprophet <interact file (with probs)>

runs analysis on inputfile using default options and writes analysis to inputfile-prot.htm

example
runprophet interact.xml
writes output to file: interact-prot.shtml
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usage 3: specify output file
runprophet (-Ooptions) <interact file (with probs)> <outputfile>

runs analysis on inputfile (with specified options) and writes analysis to specified outputfile

example
runprophet interact.xml protein.shtml
writes output to file: protein.shtml
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usage 4: combine multiple datasets into a single analysis
runprophet -Om(options) <interactfile1 interactfile2 ...> <outputfile>

runs analysis on multiple inputfiles (with specified options) and writes analysis to specified outputfile

example
runprophet -Oim interact-1.xml interact-2.xml protein.shtml
analyzes interact-1.xml and interact-2.xml icat data together and writes output to file
protein.shtml
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usage 5: options for static html output
example
runprophet -OHP0.9 interact.xml
writes results to static html file, and results with min prob 0.9 (including peptides) to tab delimited excel file
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Author

Much of this text comes from the TPP README file, originally written by A. Keller, 2004. Wiki version created by J. Tasman.

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Credits

The refreshparser program uses the SPARE Parts by Bruce W. Watson / Loek Cleophas.

Retrieved from "http://tools.proteomecenter.org/wiki/index.php?title=TPP:Developer_Documentation"
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