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    <h2>Crux Frequently Asked Questions</h2>

    <ol>

    <li>
    <b>What is the difference between Tide and Comet?</b></li>

    <p>
    The differences are as follows:
    <ul>
    <li>
    Tide converts your FASTA file to an index (either as part
    of <code>tide-search</code> or by using <code>tide-index</code>),
    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.
    <li>
    For each match, Comet reports the <i>total</i> number of candidate
    peptides, whereas Tide reports the number of <i>distinct</i> candidate
    peptides.</li>
    <li>
    Comet provides an option to report an E-value in addition to the raw
    XCorr score, whereas Tide provides options to report a variety of
    different types of scores.  The methods for computing Tide's
    various scores are described in the documentation
    for <code>tide-index</code>.</li>
    <li>
    Some options are available only in Comet (various options related to
    different types of theoretical fragment ions, max_fragment_charge,
    nucleotide_reading_frame, etc.) whereas others are only available in
    Tide (e.g., keep-terminal-aminos).</li>
    </ul></p>

    <p>
    Overall, for a given set of spectra, the XCorr scores computed by
    Comet and by Tide should be quite similar to one another, assuming
    that the various search parameters are set similarly between the two
    algorithms.  Below is a table summarizing the correspondence between
    the two sets of parameters.  The specified values will yield nearly
    identical XCorr scores from the two search engines.</p>

    <table border="1">
    <tr>
    <td>
    <b>Tide parameter</b></td>
    <td>
    <b>Tide value</b></td>
    <td>
    <b>Comet parameter</b></td>
    <td>
    <b>Comet value</b></td>
    </tr>

    <tr>
    <td>
    enzyme</td>
    <td>
    trypsin</td>
    <td>
    search_enzyme_number</td>
    <td>
    1</td>
    </tr>

    <tr>
    <td>
    digestion</td>
    <td>
    full-digest</td>
    <td>
    num_enzyme_termini</td>
    <td>
    2</td>
    </tr>

    <tr>
    <td>
    missed-cleavages</td>
    <td>
    0</td>
    <td>
    allowed_missed_cleavage</td>
    <td>
    0</td>
    </tr>

    <tr>
    <td>
    min-peaks</td>
    <td>
    10</td>
    <td>
    minimum_peaks</td>
    <td>
    10</td>
    </tr>

    <tr>
    <td>
    precursor-window</td>
    <td>
    3</td>
    <td>
    peptide_mass_tolerance</td>
    <td>
    3</td>
    </tr>

    <tr>
    <td>
    precursor-window-type</td>
    <td>
    mass</td>
    <td>
    peptide_mass_units</td>
    <td>
    0</td>
    </tr>

    <tr>
    <td>
    fragment-mass</td>
    <td>
    mono</td>
    <td>
    mass_type_fragment</td>
    <td>
    1</td>
    </tr>

    <tr>
    <td>
    decoy-format</td>
    <td>
    peptide-reverse</td>
    <td>
    N/A</td>
    <td>
    &nbsp;</td>
    </tr>

    <tr>
    <td>
    keep-terminal-aminos</td>
    <td>
    C</td>
    <td>
    N/A</td>
    <td>
    &nbsp;</td>
    </tr>

    <tr>
    <td>
    concat</td>
    <td>
    T</td>
    <td>
    decoy_search</td>
    <td>
    1</td>
    </tr>

    <tr>
    <td>
    top-match</td>
    <td>
    5</td>
    <td>
    num_results, num_output_lines</td>
    <td>
    6, 5</td>
    </tr>

    <tr>
    <td>
    remove-precursor-peak</td>
    <td>
    T</td>
    <td>
    remove_precursor_peak</td>
    <td>
    1</td>
    </tr>

    <tr>
    <td>
    remove-precursor-tolerance</td>
    <td>
    15</td>
    <td>
    remove_precursor_tolerance</td>
    <td>
    15</td>
    </tr>

    <tr>
    <td>
    use-flanking-peaks</td>
    <td>
    F</td>
    <td>
    theoretical_fragment_ions</td>
    <td>
    1</td>
    </tr>

    <tr>
    <td>
    use-neutral-loss-peaks</td>
    <td>
    F</td>
    <td>
    use_NL_ions</td>
    <td>
    F</td>
    </tr>

    <tr>
    <td>
    mz-bin-width</td>
    <td>
    1.0005079</td>
    <td>
    fragment_bin_tol</td>
    <td>
    1.0005079</td>
    </tr>

    <tr>
    <td>
    mz-bin-offset</td>
    <td>
    0.4</td>
    <td>
    fragment_bin_offset</td>
    <td>
    0.4</td>
    </tr>

    <tr>
    <td>
    min-mass, max-mass</td>
    <td>
    200, 7200</td>
    <td>
    digest_mass_range</td>
    <td>
    200 7200</td>
    </tr>

    <tr>
    <td>
    N/A</td>
    <td>
    &nbsp;</td>
    <td>
    max_fragment_charge</td>
    <td>
    2</td>
    </tr>

    </table>

    <p>
    Here is a scatter plot of XCorr scores from a search run with the
    parameters listed above:</p>

    <img src="images/stored-xcorr.png">


    <li>
    <b>What operating systems does Crux work with?</b></li>

    <p>
    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.</p>

    <li><b>When I try to run Crux on my Linux system I get error messages like:</b><br/>
    <pre>
    /tmp/crux: /usr/lib64/libstdc++.so.6: version `CXXABI_1.3.8' not found (required by /tmp/crux)
    /tmp/crux: /usr/lib64/libstdc++.so.6: version `GLIBCXX_3.4.20' not found (required by /tmp/crux)
    /tmp/crux: /usr/lib64/libstdc++.so.6: version `GLIBCXX_3.4.21' not found (required by /tmp/crux)p
    </pre>
    <br/> Crux depends on the C and C++ runtime libraries; however,
    newer versions of the runtime libraries are not backwards
    compatible with older versions.  We build the Linux binary
    distributions for Crux using GCC 4.9.  If your Linux system dates
    from before GCC 4.9 was available, then it may have versions of
    the runtime library that are incompatible with the Crux binary
    distribution.  In this case, you might consider updating your
    operating system.  Alternatively, you can build Crux from the
    source code distribution.  To build Crux from source you'll ned to
    install the C/C++ development tools, and CMake.  This can
    conveniently be done using <code>yum</code> <br/>
    <pre>
    sudo su -
    yum group install "Development Tools"
    yum install cmake
    </pre>
    <br/>
    Then follow the instructions in the in the 
    <a href="tutorials/install.html#src_build">installation tutorial</a>.
    <br/>

    <li>
    <b>How does Crux compute the masses of peptides and peptide
      fragments?</b></li>

    <p>
    <ul>

    <li>
    Each amino acid can be characterized either by its monoisotopic mass,
    which is the mass of the most abundant isotopic form of that amino acid,
    or the average mass, which is a weighted average of the masses of all
    the isotopic forms.  By default, Crux uses average mass to calculate
    peptide masses, though this behavior can be controlled by the
    <code>isotopic-mass</code> option to <code>tide-index</code> and the
    <code>mass_type_parent</code> option to <code>comet</code>.  For
    fragments, <code>tide-search</code> always uses the monoisotopic mass,
    whereas <code>comet</code> allows selection
    via <code>mass_type_fragment</code>.</li>

    <li>
    The neutral mass of a peptide is not the sum of the masses of its
    amino acids.  The N-terminus and C-terminus of the peptide together
    contribute an additional water molecule, whose mass is either
    18.010564684 Da (monoisotopic) or 18.0153 Da (average).</li>
    <li>
    A charged peptide has an additional approximately 1 Da mass for each
    charge, corresponding to the mass of a hydrogen atom.  The exact mass
    to be added depends on whether we are using the monoisotopic mass
    (1.007825035) or average mass (1.00794).</li>

    <li>When a peptide fragments, each b-ion will have a mass equal to
    the sum of its amino acids plus one hydrogen for each charge on the
    fragment, while each y-ion will have a mass equal to the sum of its
    amino acids plus water plus one hydrogen for each charge on the
    fragment.</li>

    <li>
    By default, both Tide and Comet add a static modification of
    57.021464 Da to all cysteines.  This is because in most protein
    preparation protocols the peptides are alkylated with iodoacetamide,
    resulting in carbamidomethylation of cysteine. The alkylation step
    prevents the reformation of disulfide bonds.  Other alykylation
    reagents may be used, in which case the appropriate mass shift can be
    specified with the <code>C=[mass]</code> option
    to <code>tide-index</code> or the <code>add_C_cysteine</code> option
    to <code>comet</code>.</li>

    </ul>
    </p>

    <li>
    <b>How does Crux assign fragment masses to bins prior to computing
    the XCorr score?</b>
    </li>

    <p>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, <code>mz-bin-width</code> and
    <code>mz-bin-offset</code>, control the size and location of the bins,
    and are used to convert fragment masses according to this formula:</p>

    <p align=center>binned mass = floor( ( original mass / <code>bin-width</code> ) + 1.0 - <code>bin-offset</code> )</p>

    <p>
    The default values <code>bin-width</code>=0.02
    and <code>bin-offset</code>=0.40 are suitable for most high-resolution
    datasets.
    </p>

    <li>
    <b>How can I use the average target-decoy competition (aTDC)
      procedure described in this paper?</b>
      <blockquote>
      Uri Keich, Kaipo Tamura, and William Stafford Noble.
      <a href="https://pubs.acs.org/doi/10.1021/acs.jproteome.8b00802">"Averaging
  strategy to reduce variability in target-decoy estimates of false
  discovery rate."</a> <i>Journal of Proteome Research</i>,
  18(2):585-593, 2019.
      </blockquote>
      </li>

    <ul>
      <br>
        When you build your peptide index using tide-index, you have
        to specify the number of decoy databases to include and you
        have to set allow-dups to be T. It's also important that you
        leave the decoy-format option at its default value of shuffle,
        since if you reverse the peptides then all of the databases
        would be identical.  For example, to build an
        index with five decoys per target from a fasta file
        called <code>human.fasta</code>, you could do this:<br>

<pre>
crux tide-index --num-decoys-per-target 5 --allow-dups T human.fasta human
</pre>

<br>
Also, when you search a file of spectra (<code>HepG2_f4.mzML</code>)
using the <code>human</code> index, you must specify concat=F:<br>

<pre>
  crux tide-search --use-tailor-calibration T --concat F HepG2_f4.mzML human
</pre>

<br>
This command stores the search results in the default output directory
(<code>crux-output</code>) in files
called <code>tide-search.target.txt</code>
and <code>tide-search.decoy.txt</code>. Notably, the decoy file will
contain five times as many PSMs as the target file, with a column
called "decoy index" to indicate which database the decoy comes from.
Finally, you can run aTDC by calling <code>assign-confidence</code>.
This command will use the "decoy index" column to do the necessary
averaging over the decoy databases:<br>

<pre>
  crux assign-confidence --score "tailor score" crux-output/tide-search.target.txt
</pre>
<br>
The final results are stored in a file
called <code>crux-output/assign-confidence.target.psms.txt</code>.
</ul>


    <li>
    <b>For what settings of the score-function and mz-bin-width parameter
    can I set exact-p-value=T?</b>
	<ul>
		<li>When the combined p-value score function is used (<code>--score-function=both</code>), <code>--mz-bin-width=1.0005079</code> and <code>--exact-p-value=T</code>.</li>
		<li>When the XCorr score function is used (<code>--score-function=xcorr</code>), <code>--exact-p-value</code> can only be set to true when <code>--mz-bin-width=1.0005079</code>.</li>
		<li>When the residue-evidence score function is used (<code>--score-function=residue-evidence</code>), <code>--exact-p-value</code> can only be set to true when <code>--mz-bin-width=1.0005079</code>. Note that high-resolution information in the residue-evidence score function is captured by <code>--fragment-tolerance</code>.</li>
	</ul>
    </li>

    <li>
    <b>How does Crux estimate the possible charge states of the peptides when the
       information is not provided within the spectra file?</b>
    </li>

    <p>
    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.</p>

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

    <li>
    <b>How does Crux create peptides from the given set of proteins in the
      database?</b></li>

    <p>
    The options <code>enzyme</code> (for <code>tide-index</code>)
    and <code>search_enzyme_number</code> (for <code>comet</code>) define
    the enzymatic cleavage rules.  When <code>enzyme=no-enzyme</code>
    or <code>search_enzyme_number=0</code>, 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.</p>

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

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


    <li>
    <b>How does Crux select candidate peptides from the database?</b></li>

    <p>
    The Crux search tools (<code>tide-index</code> and <code>comet</code>)
    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
    <code>precursor-window-type</code> and <code>precursor-window</code>
    options.</p>

    <ul>
    <li>  
    If the <code>precursor-window-type</code> is set to <code>mz</code>,
    then the window is calculated as spectrum precursor m/z &plusmn;
    <code>precursor-window</code> and the resulting range is converted
    to mass using the charge state with the formula:<br>  
    Mass=m/z * charge - MASS_PROTON * charge, where MASS_PROTON=1.00727646677
    </li>

    <li>
    If the <code>precursor-window-type</code> is set to <code>mass</code>, then
    the window is defined as the precursor mass &plusmn; 
    <code>precursor-window</code>.
    </li>

    <li>
    If the <code>precursor-window-type</code> is set to <code>ppm</code>
    (parts per million), then the window spans from the precursor mass /
    (1.0 + window * 1e-6) to precursor mass / (1.0 - window * 1e-6).</li>
    </ul>

    <p>
    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. </p>

    <li>
    <b>How do Tide and Comet handle combinations of static and
    variable modifications, as well as terminal
    modifications?</b></li>

    <p>
    Although the syntax for specifying amino acid modifications
    differs between Comet and Tide, the logic of how those
    modifications are applied is similar.</p>

    <p>
    First, it's important to understand that the search tools support
    several different types of modifications.  A static modification
    is one that applies to every occurrence of an amino acid, whereas
    a variable modification means that the amino acid can occur in
    either form (modified or unmodified). Furthermore, unlike standard
    modifications that occur on amino acid side-chains, a terminal
    modification is applied to the molecular group at the N- or
    C-terminus of a peptide or protein. Typically, protein terminal
    modifications correspond to biological events, whereas peptide
    terminal modifications (which by definition must be applied after
    enzymatic digestion) are produced by the experimental
    workflow.</p>

    <p>
    Conceptually, all static modifications (standard ones and terminal ones) 
    are applied first. Thereafter, variable modifications are applied on top 
    of static modifications. Tide applies at most one static modification and 
    one variable modification at any location, terminal or non-terminal. Comet 
    is similar, except that modifications can also separately be applied to 
    terminal locations. For example, the peptide DEAGFK can have a variable 
    modification on the side chain of the lysine residue as well as a variable 
    modification on the peptide C-terminus.</p>

    <p>
    In practice, it is an error to (try to) specify two different static 
    modifications for the same type of residue, but perfectly legal to specify 
    multiple variable modifications for the same residue.</p>

    <p>
    In the search engine output, you will see the unmodified peptide
    (e.g., K[-16.9981]VLGHIR in Comet's "plain_peptide" column) and
    the peptide with variable modifications indicated in brackets
    (e.g., K[-16.9981]VLGHIR in Comet's "peptide" column and Tide's
    "sequence" column). The full set of modifications (including
    static modifications) is specified as a comma-separated list in
    the "modifications" column, like this:
    1_S_45.0294,1_S_45.0239_n,1_V_-16.9981.  This specification
    indicates that the first amino acid has a standard static
    modification of 45.0294 as well as a static n-terminal
    modification of 45.0239.  In addition, the same residue has a
    variable modification of -16.9981.</p>

    <p>
    Note that Comet allows slightly more flexibility than Tide in
    specifying different types of modifications.  Terminal
    modifications in Comet can be specified to occur within a range of
    amino acids near the terminus.  Also, via the "binary set" flag, a
    modification can be required to appear on all or none of the
    residues in a given peptide.</p>

    <li>
      <b>How does Tide handle peptide and protein terminal modifications?</b></li>
  
      <p>
        Peptide and protein terminal modifications are similar in their logic. 
        The <code>(c/n)term-peptide-mods-spec</code> option applies modifications to the terminals 
        of all peptides, whereas <code>(c/n)term-protein-mods-spec</code> applies modifications only 
        to peptide terminals that are also protein terminals. If both peptide and protein 
        mod specs are provided, then both will be applied. It is possible to simultaneously 
        specify peptide and protein terminal mods for the same type of residue. If both 
        are static then protein terminals will have both of them (sum of the mod masses). 
        If both are variable then they will be applied one at a time.
      </p>
      <p>
        Note that the Tide index does not store information about the origin of 
        variable mods. As a result, the Tide search output, unlike Comet, does not 
        discriminate between terminal and non-terminal mods. Thus, Tides modified 
        sequence column always has format K[-16.9981]VLGHIR, whereas in Comet the same 
        peptide would be specified as n[-16.9981]VLGHIR for a terminal mod and 
        K[-16.9981]VLGHIR for a non-terminal mod.  Similarly, the modifications 
        column in Tide search results always has the format [position]_V_[mass]. 
        Static modifications do not have this issue: they will have a _c or _n 
        suffix for peptide terminal mods and _C or _N for protein terminal mods.
      </p>
    <li>
    <b>What happens if I decrease the size of the precursor window during
    searching?</b></li>

    <p>
    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 <code>precursor-window</code> and
    <code>precursor-window-type</code> options.</p>

    <li>
    <b>How can I search my isotopically labeled data with Tide or Comet?</b></li>
    <p>
    Isotopically labeled data is accommodated by specifying various static or
    variable modifications. The specific types of modifications differ according
    to the labeling scheme, and the syntax for specifying the modifications
    differs between Tide (which uses options like <code>--mods-spec</code>) and
    Comet (which uses options like <code>add_Nterm_peptide</code>). In each of
    the following entries, we provide first the Tide options, followed by the
    Comet options.</p>
    <p style="color: #00f;">iTRAQ 4-plex:</p>
    <p><code>
    --mods-spec K+144.10253 --nterm-peptide-mods-spec X+144.10253</code></p>
    <p><code>
    add_Nterm_peptide = 144.10253<br>
    add_K_lysine = 144.10253<br>
    clear_mz_range = 113.5 117.5</code></p>
    <p>
    The 4-plex reagent has different  monoisotopic mass values for 114
    (144.105918), 115 (144.09599), and 116/117 (144.102063).  The mass value
    used above is derived from averaging the three monoisotopic masses.</p>
    <p style="color: #00f;">iTRAQ 8-plex:</p>
    <p><code>
    --mods-spec K+304.2022 --nterm-peptide-mods-spec X+304.2022</code></p>
    <p><code>
    add_Nterm_peptide = 304.2022<br>
    add_K_lysine = 304.2022<br>
    clear_mz_range = 112.5 121.5</code></p>
    <p>
    The mass modification above is the average of the two different set of
    masses for 115/118/119/121 (304.199040) and 113/114/116/117
    (304.205360).</p>
    <p style="color: #00f;">TMT 2-plex:</code></p>
    <p><code>
    --mods-spec K+225.155833 --nterm-peptide-mods-spec X+225.155833</code></p>
    <p><code>
    add_Nterm_peptide = 225.155833<br>
    add_K_lysine = 225.155833<br>
    clear_mz_range = 125.5 127.5</code></p>
    <p style="color: #00f;">TMT 6-plex:</code></p>
    <p><code>
    --mods-spec K+229.162932 --nterm-peptide-mods-spec X+229.162932</code></p>
    <p><code>
    add_Nterm_peptide = 229.162932<br>
    add_K_lysine = 229.162932<br>
    clear_mz_range = 127.5 131.5</code></p>
    <p style="color: #00f;">SILAC 4Da:</p>
    <p>
    There are a number of different SILAC reagents with a ~4 Da modification
    (based on combinations of C13 and N15), each with different sites of
    specificity.  The example below is for the 15N(4) reagent applied to R
    residues.  You should adjust the modification mass and residue(s) applied
    to as necessary.</p>
    <p>
    To perform a mixed light/heavy search using a variable modification
    search:</p>
    <p><code>
    --mods-spec 3R+3.988140</code></p>
    <p><code>
    variable_mod01 = 3.988140 R 1 3 -1 0 0</code></p>
    <p>
    Note that, in the example above, Comet operates in “binary” mode, which
    means that it only considers peptides in which all or none of the lysine
    residues are light/heavy. Tide does not support binary mode searching, so it
    will consider all peptides with up to 3 (in the above specification) heavy
    lysines.</p>
    <p>
    To search just the heavy labeled sample, you can apply a static
    modification:</p>
    <p><code>
    --mods-spec R+3.988140</code></p>
    <p><code>
    add_K_lysine = 3.988140</code></p>
    <p style="color: #00f;">SILAC 6Da:</p>
    <p>
    This example uses the 13C(6) SILAC mass, assuming it is applied to both K
    and R. You should adjust as necessary.  There is at least one more SILAC
    reagent with ~6 Da modification mass and different residue specificity:
    13C(5) 15N(1)</p>
    <p>
    To perform a mixed light/heavy search using a variable modification:</p>
    <p><code>
    --mods-spec 3KR+6.020129</code></p>
    <p><code>
    variable_mod01 = 6.020129 KR 1 3 -1 0 0</code></p>
    <p>
    To search just the heavy labeled sample, you can apply a static
    modification:</p>
    <p><code>
    --mods-spec KR+6.020129</code></p>
    <p><code>
    add_K_lysine = 6.020129<br>
    add_R_arginine = 6.020129</code></p>
    <p style="color: #00f;">SILAC 8Da:</p>
    <p>
    The example below is for 13C(6) 15N(2) on K residues. Variable modification
    search:</p>
    <p><code>
    --mods-spec 3K+8.014199</code></p>
    <p><code>
    variable_mod01 = 8.014199 K 1 3 -1 0 0</code></p>
    <p>
    Static modification for just the heavy labeled search:</p>
    <p><code>
    --mods-spec K+8.014199</code></p>
    <p><code>
    add_K_lysine = 8.014199</code></p>


<p style="color: #00f;">100% 15N label:</p>

<p>
  The N15 labeling adds 0.997035 mass difference for every nitrogen in
  each amino acid.  So for a 100% N15 analysis, specify a static
  modification (between 1 and 4) to every amino acid residue, like
  this:</p>

<p><code>
--mods-spec A+0.997035,C+0.997035,D+0.997035,E+0.997035,F+0.997035,G+0.997035,H+2.99111,I+0.997035,K+1.99407,L+0.997035,M+0.997035,N+1.99407,P+0.997035,Q+1.99407,R+3.98814,S+0.997035,T+0.997035,V+0.997035,W+1.99407,Y+0.997035</code></p>

<p>
<pre>
add_G_glycine = 0.997035               # added to G - avg.  57.0513, mono.  57.02146
add_A_alanine = 0.997035               # added to A - avg.  71.0779, mono.  71.03711
add_S_serine = 0.997035                # added to S - avg.  87.0773, mono.  87.03203
add_P_proline = 0.997035               # added to P - avg.  97.1152, mono.  97.05276
add_V_valine = 0.997035                # added to V - avg.  99.1311, mono.  99.06841
add_T_threonine = 0.997035             # added to T - avg. 101.1038, mono. 101.04768
add_C_cysteine = 0.997035              # added to C - avg. 103.1429, mono. 103.00918
add_L_leucine = 0.997035               # added to L - avg. 113.1576, mono. 113.08406
add_I_isoleucine = 0.997035            # added to I - avg. 113.1576, mono. 113.08406
add_N_asparagine = 1.99407             # added to N - avg. 114.1026, mono. 114.04293
add_D_aspartic_acid = 0.997035         # added to D - avg. 115.0874, mono. 115.02694
add_Q_glutamine = 1.99407              # added to Q - avg. 128.1292, mono. 128.05858
add_K_lysine = 1.99407                 # added to K - avg. 128.1723, mono. 128.09496
add_E_glutamic_acid = 0.997035         # added to E - avg. 129.1140, mono. 129.04259
add_M_methionine = 0.997035            # added to M - avg. 131.1961, mono. 131.04048
add_O_ornithine = 1.99407              # added to O - avg. 132.1610, mono  132.08988
add_H_histidine = 2.99111              # added to H - avg. 137.1393, mono. 137.05891
add_F_phenylalanine = 0.997035         # added to F - avg. 147.1739, mono. 147.06841
add_U_selenocysteine = 0.997035        # added to U - avg. 150.3079, mono. 150.95363
add_R_arginine = 3.98814               # added to R - avg. 156.1857, mono. 156.10111
add_Y_tyrosine = 0.997035              # added to Y - avg. 163.0633, mono. 163.06333
add_W_tryptophan = 1.99407             # added to W - avg. 186.0793, mono. 186.07931
add_B_user_amino_acid = 0.0000         # added to B - avg.   0.0000, mono.   0.00000
add_J_user_amino_acid = 0.0000         # added to J - avg.   0.0000, mono.   0.00000
add_X_user_amino_acid = 0.0000         # added to X - avg.   0.0000, mono.   0.00000
add_Z_user_amino_acid = 0.0000         # added to Z - avg.   0.0000, mono.   0.00000
</pre>    

<p>
  If you need to adjust the cysteine mass to account for
  carboxyamidomethylation, then use 58.018499 instead of 0.997035 for
  C. </p>

<p>
<code>
--mods-spec A+0.997035,C+58.018499,D+0.997035,E+0.997035,F+0.997035,G+0.997035,H+2.99111,I+0.997035,K+1.99407,L+0.997035,M+0.997035,N+1.99407,P+0.997035,Q+1.99407,R+3.98814,S+0.997035,T+0.997035,V+0.997035,W+1.99407,Y+0.997035</code></p>

<p><pre>
add_C_cysteine = 0.997035
</pre></p>

    <li>
    <b>How can I contribute to Crux?</b></li>

    <p>
    Patches implementing new features can submitted directly as pull
    requests on github, or can be be emailed to the development
    team at crux-support@uw.edu for review and inclusion in subsequent
    releases of Crux.</p>

    <li>
    <b>Where does the name "Crux" come from?</b></li>

    <p>
    Thin air.  The name is not an acronym or a reference to anything in
    particular.</p>

    </ol>
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