Crux Frequently Asked Questions

  1. What is the difference between Tide and Comet?
  2. The main difference is that Tide requires that you convert your FASTA file to an index (using tide-index) before doing a search (using tide-search), whereas Comet does searching directly from the FASTA file. The indexing step allows Tide to pre-compute many parts of the search procedure, thereby making the search faster. Other differences are as follows:

  3. What operating systems does Crux work with?
  4. Crux is written in C++, so in principle it should work on virtually any modern operating system. We provide pre-compiled binaries for Linux, MacOS and Windows.

  5. How does Crux compute the masses of peptides and peptide fragments?
  6. How does Crux assign fragment masses to bins prior to computing the XCorr score?
  7. The XCorr score is essentially a dot product between a preprocessed form of the observed spectrum and the theoretical spectrum derived from the candidate peptide. In order to compute this dot product, the masses of the fragments in both spectra are assigned to discrete mass bins. This can be viewed as a form of rounding, but with more control over the discrete masses. Two parameters, mz-bin-width and mz-bin-offset, control the size and location of the bins, and are used to convert fragment masses according to this formula:

    binned mass = floor( ( original mass / bin-width ) + 1.0 - bin-offset )

    The default values bin-width=1.0005079 and bin-offset=0.40 are suitable for most low-resolution datasets.

  8. How does Crux estimate the possible charge states of the peptides when the information is not provided within the spectra file?
  9. Crux calculates the ratio of two values: (1) the sum of the intensities from the peaks in the fragmentation spectrum whose m/z is greater than the precursor m/z versus (2) the sum of the peaks whose m/z is smaller than the precursor. If this ratio is greater than or equal to a calculated threshold based upon the location of the precursor m/z and the max m/z in the spectrum, Crux then assigns both +2 and +3 as possible charge states to the spectrum. Otherwise, Crux assigns +1 as the charge state. The algorithm is based upon the observation that fragmentation spectra collected from +1 peptides should have no peaks above the precursor m/z. In contrast, a peptide of charge state greater than +1 can generate fragment ions of lower charge whose m/z is greater than the precursor m/z, thus indicating a multiply charged precursor.

    Note that, in addition, comet includes a precursor_charge parameter. If the first number in this parameter is set to 0, i.e., precursor_charge = 0 0, then the charge state rule above is applied. However, if a user specifies a precursor charge range, i.e., precursor_charge = 1 5, then Comet will search every spectrum through this range of assumed charge states for every spectrum whose precursor charge is unknown.

  10. How does Crux create peptides from the given set of proteins in the database?
  11. The options enzyme (for tide-index) and search_enzyme_number (for comet) define the enzymatic cleavage rules. When enzyme=no-enzyme or search_enzyme_number=0, then any subsequence of a protein may be considered as a candidate peptide. For other values of these parameters, the residues at the termini of the protein subsequence must follow specific rules. For example, trypsin requires that the preceeding residue must be an R or K and the following residue may not be a P.

    When digestion=full-digest (for tide-index) or num_enzyme_termini=2 (for comet), then these rules must be true at both ends of the peptide. When it is digestion=partial-digest or num_enzyme_termini=1, then the rules must be true at at least one of the ends. The missed-cleavages and allowed_missed_cleavages parameters control the maximum number of cleavage sites that may lie within the peptide sequence.

    Note that if enzyme=no-enzyme or search_enzyme_number=0, then the digestion, num_enzyme_termini, missed-cleavages and allowed_missed_cleaveages parameters are not used.

  12. How does Crux select candidate peptides from the database?
  13. The Crux search tools (tide-index and comet) select candidate peptides for each spectrum based on its precursor singly charged mass (m+h) or the mass-to-charge (m/z) and an assumed charge state (specified in the input file). If the m+h and charge is provided in the input file (e.g., from the Z lines of an MS2 file), then the precursor mass is calculated from the m+h minus the mass of a proton. Otherwise, the precursor mass is calculated from the precursor m/z and an assumed charge. A mass window is defined in one of three ways based on the precursor-window-type and precursor-window options.

    Candidate peptides are those whose mass falls within the defined window. The peptide mass is computed as the sum of the average amino acid masses plus 18 Da for the terminal OH group. Candidate peptides can also be constrained by minimum and maximum allowed length.

  14. How can I search my ITRAQ data?
  15. ITRAQ and other tagged seaches can be done by using terninal modifications. For example, if your tag has a mass of 304.199040, add these lines to your parameter file:

    nmod=304.199040:-1
    cmod=304.199040:-1
    

    Crux will then generate peptides with this modification (+304.199904) on the n-terminus, the c-terminus and on both termini. The -1 in the option means to add the modification regardless of where the peptide terminus lies relative to the rest of the protein.

  16. What happens if I decrease the size of the precursor window during searching?
  17. Reducing the precursor mass tolerance has two main benefits. One is reduced search time, and the other is improved statistical power to detect matches. With a smaller window, fewer candidates are tested against a spectrum. As a result, the statistical confidence measure calculated after multiple testing correction will be more significant. Of course, the flipside is that if you make the precursor window too small, then you may end up throwing out correct identifications. Control over the size of the window is provided by the using the precursor-window and precursor-window-type options.

  18. How can I contribute to Crux?
  19. Patches implementing new features can be emailed to the development team at crux-support@uw.edu for review and inclusion in subsequent releases of Crux.

  20. Where does the name "Crux" come from?
  21. Thin air. The name is not an acronym or a reference to anything in particular.


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