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Spectral Library Preparation from DDA Data
Warning: This is a Rough Draft
Using your raw mass spectrometry data, run a database search with the output in pepXML format. We used fragpipe in MS-Fragger, and recommend you follow their instructions.
Spectrast is part of the Trans Proteomic Pipeline (TPP) suite. You will need to download the suite to run Spectrast - see here for instructions on downloading the suite.
Below are the command line commands we used to compile our final library. For explanations or instructions for tailoring the output to specific parameters, please look at the command options for spectrast (run \<path to TPP folder\>TPP/bin/spectrast.exe -h at the command line).
<path to TPP folder>TPP/bin/spectrast.exe -M spectrast.usermods -c_BIN! -c_RDYrev_ -cP0.9584 -cICID -cN <list pep.xml files>
**Note: the mass spectrometry data (such as .mzML or .mzXML files) corresponding to the pep.xml files needs to be in the same folder as them.
<path to TPP folder>TPP/bin/spectrast.exe -M spectrast.usermods -cAC -c_BIN! -cICID -cN <path to desired output file>outputcid_con <.splib file output from previous command>
<path to TPP folder>TPP/bin/spectrast.exe -M spectrast.usermods -cAD -cc -c_BIN! -cICID -cN <path to desired output file>consensus_plus_decoys <path to output from last command>outputcid_con.splib
You will need to download the msproteomicstools package from Github. Instructions for download are in the Readme. Note that, in accordance with our paper, we prepared the paper with
python <path to spectrast2tsv.py in msproteomicstools package>spectrast2tsv.py -m <path to spectrast.usermods output from ms-fragger>spectrast.usermods -l 400,2000 -s b,y -x 1,2 -o 3 -n 20 -p 0.2 -d -e -k openswath -a <path to desired output file>consensus_transitions_lowres_decoys.tsv <path to output from last command>consensus_plus_decoys.splib