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 paper by Atwood and York http://www.peptideatlas.org/upload/Atwood-York_GlycopeptideSearchStrategy.pdf, but has been modified to work with the ISB version of Sequest.

  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

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