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Preparing MSFragger
Mass spectrometry data must first be converted to one of the supported MS/MS input formats of MGF, mzXML, or mzML. A popular option for converting from vendor file inputs and between various input formats is Proteowizard (proteowizard.sourceforge.net). MSFragger determines the appropriate data parser to use based on the file extension (.mgf for MGF, .mzXML for mzXML, and .mzML for mzML) and does not make inferences from file contents (i.e. naming a mzML file with the .mzXML extension will lead to unpredictable results or crashes). Note that there is extremely limited support for MGF files so it is recommended that they first be converted to mzML prior to search.
Extract the MSFragger.jar into your working directory along with the sample configuration file called fragger.params. MSFragger is configured using a text parameters file. The parameters file is passed as the first argument to MSFragger and has no restrictions on names or file extensions (so one might want to name their configuration files to be more descriptive such as Uniprot_open_withmods.txt) after editing the parameters file for a particular analysis.
Parameter names are given left of the equal sign and parameter values are given to the right (e.g. num_threads = 4). White spaces are trimmed from the ends of each value by MSFragger. All text to the right of (and including) the # sign of each line is discarded so # can be used for comments in the parameters file.
MSFragger allows multiple MS/MS input files to be processed in a batch. Passing multiple files to MSFragger at once allows MSFragger to reuse the fragment index for subsequent MS/MS run. This is particularly important for narrow window searches which may only take fractions of a second.
On computers or compute clusters with many processor cores, we highly recommended that MSFragger is set to process files sequentially with all available processor cores rather than running multiple instances of MSFragger in parallel (assigning a smaller number of cores to each). This reduces initialization times and allows the fragment index to be re-used, at the same time reducing overall memory requirements.