Alt NxST search strategy

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-	The instructions below are for searching MS/MS data from glycocapture experiments. It is based on a 2005 <A HREF="http://www.peptideatlas.org/upload/Atwood-York_GlycopeptideSearchStrategy.pdf"> paper by Atwood and York </A>, but has been modified to work with the ISB version of Sequest.	+	The instructions below are for searching MS/MS data from glycocapture experiments. It is based on a 2005 http://www.peptideatlas.org/upload/Atwood-York_GlycopeptideSearchStrategy.pdf paper by Atwood and York </A>, but has been modified to work with the ISB version of Sequest.
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Revision as of 22:25, 13 November 2008

The instructions below are for searching MS/MS data from glycocapture experiments. It is based on a 2005 http://www.peptideatlas.org/upload/Atwood-York_GlycopeptideSearchStrategy.pdf paper by Atwood and York </A>, but has been modified to work with the ISB version of Sequest.

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1. All work should generally be done as user sbeams

su sbeams

1. set path to include scripts, bash syntax shown

export PATH=$PATH:/regis/sbeams/archive/sbeams/scripts

1. create search database with NxS/T changed to NBS/T. This will
2. make nxst and 'reverse' versions of the source database, called
3. myfasta_nxst_db.fsa myfasta_reverse_db.fsa for the example shown.

make_nxst_db.pl myfasta.fsa

1. Make search directory

mkdir HsIPI_v3.30plus_LTQ_nxst cd HsIPI_v3.30plus_LTQ_nxst

1. Copy in template sequest.params file, which has static mass mod for B.
2. Edit database to point at modified db

cp /regis/sbeams/archive/sbeams/conf/sequest.params.ft . mv sequest.params.ft sequest.params

1. Add symlinks to mzXML files

ln -s /data2/search/yzhou/CP/LCQ/Prostate_*.mzXML .

1. Run sequest

runsequest *.mzXML

1. Generate xml from html results

ls *html | xargs batch_sequest_2_xml.sh

1. Substitute search settings

ls *.pep.xml | xargs batch_convert.sh

1. This step is still manual, substitute db name in pepXML files, as well as
2. copy of sequest.params file

cp sequest.params sequest.params.run perl -pi -e 's/ipi.MOUSE.v3.24.fasta-mix7prot_nxst_db.fsa/ipi.MOUSE.v3.24.fasta-mix7prot.fsa/' *.xml sequest.params

1. Create or copy over an appopriate xinteract command

cp /regis/sbeams/archive/sbeams/conf/xinteract.cmd.ft xinteract.cmd

1. Run xinteract

./xinteract.cmd

1. Run calctppstat.pl script to see results

calctppstat.pl --fu -g