After you have run a search and
assigned confidence to your identified PSMs, you can now calculate
relative quantification values for the proteins by running the
$ crux spectral-counts --threshold 0.1 --protein-database small-yeast.fasta ./crux-output/percolator.target.psms.txt
While the command is running, you will see output like this
INFO: CPU: guanine.gs.washington.edu INFO: Crux version: 4.0-ad795d65-2021-08-23 INFO: Tue Sep 14 21:35:02 PDT 2021 INFO: Beginning spectral-counts. INFO: Total proteins found: 56 INFO: Assigning index 0 to ./crux-output/percolator.target.psms.txt. INFO: Number of matches:9134 INFO: Number of matches passed the threshold 1515 INFO: Number of peptides 758 INFO: Number of proteins 54 INFO: Elapsed time: 0.263 s INFO: Finished crux spectral-counts. INFO: Return Code:0
The crux-output directory will now contain three new files:
- spectral-counts.target.txt – tab-delimited file containing the
- spectral-counts.params.txt – parameter file
- spectral-counts.log.txt – log file.
The spectral-counts.target.txt file will look something like:
protein id NSAF YGR192C 0.14454275 YCR012W 0.07278429 YBR118W 0.070475481 YLL024C 0.045148738 YOR063W 0.045023989 YDR064W 0.039725326 YLR043C 0.027732428 YLR249W 0.027634142
In this output, the first field is the identifier and the second field is the spectral-counting measure (NSAF, dNSAF, SIN, or EMPAI) result for each protein. To select which measure is computed, see the crux spectral-counts documentation.
Using peptideprophet probability from pepXML
crux spectral-counts also supports
using Peptide Prophet probabilities provided in a
pepXML file. Use the command:
$ crux spectral-counts --protein-database small-yeast.fasta --threshold-type custom --custom-threshold-name peptideprophet --threshold 0.9 --custom-threshold-min F target.pep.xml
Using mzIdentML files with spectral-counts
crux spectral-counts also supports mzIdentML as input. For example,
If the protein sequences are not provided within the given mzIdentML file, then you will have to provide a database using the protein-database parameter for the NSAF, dNSAF, EMPAI, and SIN metrics.