## Release notes for Crux

### Version 5.0.0

April 15, 2026

#### Major changes

- 15 April 2026: Added new command `crux lfq` for label-free quantification (LFQ). The command takes a set of peptide-spectrum matches (PSMs) and corresponding spectrum files and quantifies each detected peptide at the MS1 level. The algorithm follows that of FlashLFQ (Millikin et al., Journal of Proteome Research, 2018). Supports both Crux `.psmtsv` and Percolator output formats, optional intensity normalization, and protein-level quantification via median polish.

### Version 4.3.2

April 3, 2026

#### Minor changes

- 10 March 2026: Fixed name of comet target output file when using separate search.
- 10 March 2026: Updated to 2026.01.0 release of Comet.
- 10 March 2026: Fixed linked libraries in debug build.
- 17 Sept 2025: Bug fix in --store-spectra T .
- 27 Aug 2025: Bug fix in output results format conversion in tide-search.

### Version 4.3.1

August 7, 2025

#### Major changes

- 23 July 2025: Updated to ProteoWizard 3.0.25123.b0e5f51.
- 19 March 2025: Updated to use Comet 2025.01.1, including support for indexing a sequence database.

#### Minor changes

- 27 Aug 2025: Bug fix in output results format conversion in tide-search.
- 23 July 2025: Updated GitHub build actions to use windows-latest and added macos-15.
- 23 July 2025: Updated build files to support Visual Studio 2022 and later.
- 23 July 2025: Update `crux comet` parameter default to match Comet 2025.01.1.
- 10 June 2025: Updated pepXML format.
- 17 April 2025: Fixed a bug in reporting tide-search results in various output formats.

### Version 4.3

March 25, 2025

#### Major changes

- 19 March 2025: Updated to use Comet 2025.01.1, including support for indexing a sequence database.
- 11 December 2024: Updated to use Percolator rel 3-07-01. Retention time is now included in the Percolator output.
- 18 Oct 2024: Added support for tide-search to produce output in mztab format.
- 18 Oct 2024: Added tide-index support for unimod codes.

#### Minor changes

- 6 Feb 2025: Created the spectrum-convert application.
- 28 Jan 2025: Bug fix in tide-search when searching without decoy peptides.
- 25 Oct 2024: Fixed bug in parameter handling for spectral-counts inside of the pipeline command.
- 25 Oct 2024: Added .gz format as read fasta file option.
- 22 Oct 2024: Added error message when user specifies non-supported score functions. Fixed a bug that prevented building on windows and mac.
- 18 Oct 2024: Terminal amino acids can now be modified by terminal modifications and resiudal mods.
- 18 Oct 2024: Tide-search reports tailor scores and statistics about b-y- ions.
- 18 Oct 2024: Tide-search runs only combined p-values.
- 18 Oct 2024: Unused options removed from tide-search: --compute-sp, --file-column, --brief-output, --peptide-centric-search, --evidence-granularity, --use-tailor-calibration (it is automatically calculated), From tide-index:--keep-terminal-aminos.
- 15 September 2024: Unify sorting, replacing quicksort algorithm with std::sort
- 11 September 2024: Removed usage plot from Crux website home page.
- 30 July 2024: Modified tool use events to submit via GA4.
- 02 July 2024: Typo fix in the documentation of precursor window calculation.

### Version 4.2

April 6, 2024

#### Major changes

- 6 April 2024: Updated to use Percolator commit d0d48b5.
- 11 March 2024: Updated to use ProteoWizard 3.0.24044.fd6604. This required shifting Crux and dependencies to the C++14 standard and disabling the C++11 ABI. This fixed issues building Crux on newer releases of Linux and MacOS. The Centos 7 build was replaced with Ubuntu 20.04 in GitHub commit action for Master branch. The recommended version of CMake is now 3.24. Earlier versions will work, but you may see some warning messages.

#### Minor changes

- 12 April 2024: Fixed bug in ArgParser with repeated options and arguments.
- 6 April 2024: Updated UNIMOD definitions.
- 26 March 2024: Updated smoke tests.
- 11 Jan 2024: added spectral-counts to pipeline command.
- 11 Jan 2024: Fixed bug where NSAF runs to completion with no database.
- 14 Nov 2023: Fixed bug in spectral-counts documentation, LaTex not being rendered.
- 10 Oct 2023: When a parameter introduced by user doesn't match, app halts with an error.
- 5 Oct 2023: Fixed bug in Bullseye and Hardklor which prevented them from processing files with a '#' in the file name.
- 1 Oct 2023: Removed uneeded DLL from Windows distribution. Fixed error in Thermo RAW file support.
- 1 Sept 2023: Fixed several bugs related to spectral counts. Spectral counts can now parse native Percolator output.
- 25 July 2023: crux percolator --pepxml-output T now uses internal percolator code to generate pepxml output.
- 10 July 2023: Added support for the percolator --no-terminate option, which allows execution to continue even if the SVM encounters issues.
- 30 May 2023: File overwrite bug fix in diameter
- 14 April 2023: Added by-ions matched, by-ions total, and by-ions-fraction columns to tide-search output in case of standard XCorr scoring.
- 14 March 2023: Added a new column, called "unmodified sequence," to tab-delimited outputs. This column contain the peptide sequence without modifications. The new column appears in tide-search, diameter, assign-confidence, and cascade-search results.
- 14 March 2023: The "cleavage type" column was removed from tab-delimited outputs produced by tide-search, diameter, and assign-confidence.
- 20 Feb 2023: Tide-index aux locations were merged into the peptide protocol buffer.
- 20 Feb 2023: Tide-index now shuffles modified and unmodified target peptides in the same way.
- 19 Feb 2023: Add a "filename" column to the Percolator tab-delimited output files.
- 07 Feb 2023: Fix bug in mix-max procedure. The q-values are now the smallest FDRs for the associated thresholds.
- 13 Jan 2023: Tide-search halts with an error message when the user tries to use tailor-calibration in conjunction with peptide-centric search.
- 13 Jan 2023: Added a check to verify that these release notes are updated in every pull request.
- 09 Jan 2023: Upgraded to Comet 2022.01 rev 2. Fixed issue #572: handling of Comet isotope_error option (now allows values 0-5).
- 04 Jan 2023: Fix divide-by-zero bug in postprocessing diameter results.
- 16 Dec 2022: Changed the default precision for specifying variable modifications to four decimal points rather than two.
- 13 Dec 2022: Updated to Kojak commit 4307dfa from Oct 11 2022 and MSToolkit commit 20e99ce from Sept. 14 2022. Added NeoPepXMLParser commit e89f166 from Jul 1 2022 as an external dependency.
- 15 Oct 2022: The percolator command no longer creates mzIdentML output, and the tab-delimited text format produced by crux percolator now mimics that of the stand-alone version of Percolator. The crux spectral-counts and assign-confidence commands have been updated to parse the new percolator output files.Updated to percolator committ 879af86.
- 28 Sept 2022: Updated Assign-confidence to the new comet output.
- 28 Sept 2022: Updated performance and smoke tests
- 28 Sept 2022: Added option to tide-search to search spectra in a min- and max-precursor-charge range.
- 28 Sept 2022: Removed the option from tide-index to generate decoy protein fasta file.
- 28 Sept 2022: Fixed bug in tide index which prevented it generating terminal variable modifications.
- 28 Sept 2022: Fixed bug in tide index which slowed it down when generating many mods.
- 22 Aug 2022: The scalability of tide-index has been improved to allow for creation of very large peptide databases. This is done by sorting and filtering the peptides on disk rather than in memory. A new parameter, --memory-limit, allows the user to specify an upper bound on the memory (i.e. RAM) usage of tide-index. Note that this update required that the data structure used in Tide's index files has changed. It is therefore recommended that users' recreate old index files. Currently, indices in the old format can still be used with tide-index, but this format may be deprecated in future releases of Crux.
- 17 August 2022: Updated to percolator committ 1e82daa.
- 27 July 2022: Fixed bug preventing percolator from running on Kojak output.
- 19 July 2022: Added min_peptide_score parameter to Kojak.
- 09 June 2022: Upgraded to ProteoWizard 3.0.22014 (commit aadd392). This also upgrades to BOOST 1.67 which fixes a problem building Crux on current MacOS systems.
- Upgraded to Protocol Buffers 3.19.4
- Upgraded to GFlags 2.2.2
- 09 June 2022: Added support for building using GitHub Actions.
- 01 May 2022: XCorr scoring implementing in C++. This resolves a longstanding bug in our multithreading code.
- 20 Jan 2022: The XCorr scoring, exact p-value, and ResEv in Tide-Search has been sped up.
- 10 Jan 2022: Fixed bug that resulted in rank=0 for search results in pepxml files (issue #521).
- 16 Nov 2021: The preprocessing of the exact p-value method has been aligned to the preprocessing of the standard XCorr scoring.
- 16 Nov 2021: Comet has been upgraded to version 2021.01.0.
- 16 Nov 2021: Kojak has been upgraded to commit defa8c9.
- 16 Nov 2021: MSToolkit has been upgraded to commit 4cb705d.
- 16 Nov 2021: IUPAC symbol 'O' is now the amino acid pyrrolysine.
- 16 Nov 2021: Dropped support for 32-bit versions of Crux.
- 9 Nov 2021: Fix the crash bug in `diameter` when there is no MS1 spectrum in the input file.
- 5 Nov 2021: Update the param-medic documentation to include pointers to published papers and to describe detection of modifications.
- 30 Oct 2021: Remove the protein-reverse option from "--decoy-format" in `tide-index` and `generate-peptides`.
- 23 Oct 2021: Revised the `tide-index` so that "--peptide-list T" will generate a single text file listing of all the peptides in the database, along with their corresponding decoy peptides, neutral masses and proteins, one per line.
- 18 Oct 2021: Fixed a bug in DIAmeter that caused it to detect 0 PSMs when the `windowWideness` property is missing in the input file.
- 18 Oct 2021: Revised the DIAmeter documentation to clarify which options are affected by the setting of `diameter-instrument`.

### Version 4.1

October Oct. 11, 2021

#### Major changes

- 06 Oct 2021: Added DIAmeter module for searching for detecting peptides from data-independent acquisition mass spec data without requiring a spectral library. The methodology is described in
  Lu et al. Bioinformatics 37(Supplement_1):i434–i442, 2021.

#### Minor changes

- 14 Sept 2021: Add second set of smoke-tests that give tide-search a FASTA file instead of a database index.
- 14 Sept 2021: Replace contents of demo.ms2 in the tutorials with new data (bug #454).
- 10 Jun 2021: Fixed bug in computing the m/z window size in Tide. Affected only the setting when `precursor-window-type=mz`.
- 7 Jun 2021: Revised documentation and added check to ensure that the brief-output option to tide-search is not used in conjunction with formats other than tab-delimited text output.
- 11 May 2021: Fixed bug in p-value search that incorrectly set the bin_width to 0 when tide-search was given a FASTA file.
- 11 May 2021: Added smoke tests to test case where tide-search is given a FASTA file instead of a index.

### Version 4.0

April 06, 2021

#### Major changes

- Mar. 9, 2021: Removed seldom used commands: xlink, xlink-assign-ions, xlink-score-spectrum, barista, q-ranker, extract-columns, extract-rows, stat-column, sort-by-column.
- Nov. 6, 2020: Updated to use Comet 2019.05.
- Nov. 6, 2020: Added Kojak module for searching for cross-linked peptides. The methodology is described in
  Hoopmann et al. Journal of Proteome Research, 2015.
- July 23, 2019: Added the Tailor PSM score calibration method to Tide-search as an option.
- June 4, 2019: The Crux automatic build process was modified to use a cached version of the Proteowizard source, rather than always using the latest version available. The current cached version is Proteowizard 3.0, Git commit c172999. You can configure the Crux build to use the most recent available Proteowizard source code by passing the option -DUSE_LATEST_PWIZ=ON on the CMake command line.
- April 23, 2019: Updated Param-Medic to enable inference of the presence of various types of modifications, including stable-isotope and isobaric labeling and tandem mass tags as well as the enrichment of phosphorylated peptides. The methodology is described in May et al. Journal of Proteome Research, 2019.

#### Minor changes

- Mar 10, 2021: Changed num-threads default to 1.
- Feb. 12, 2021: Added date of latest commit to version string.
- Feb. 11, 2021: Fixed bug in support for comet. Output files were being stored in the wrong directory when multiple spectra files were provided.
- Feb 9, 2021: Added process ID number to the names of temporary files created by Tide, to avoid conflicts.
- Jan. 11, 2021: Fixed break in build of Percolator.
- December 7, 2020: Updated documentation to remove "+" from the usage statement.
- December 4, 2020: Updated schematic diagram of Crux tools.
- December 3, 2020: Fixed bug in peptide-centric search that caused mis-scaling of XCORR score on some Linux systems.
- September 16, 2020: Add an additional parser to parse scan number out of the “TITLE” line in MGF files. Now the TITLE line can consist of two formats. The first format is TITLE=fileName.scanNumber.scanNumber.charge.dta. The second format is TITLE=fileName.scanNumber.scanNumber.charge File:"fileNameWithExtension", NativeID:"controllerType=0 controllerNumber=1 scan=scanNumber"
- Sept. 18, 2020: Updated MGF file parser to handle title line.
- August 28, 2020: Updated documentation for --use-neutral-loss-peaks.
- August 27, 2020: Added support for protein terminal modifications. See FAQ page for details.
- August 7, 2020: Added --lysarginase as an enzyme option to tide-index and generate-peptides. The rule this enzyme follows is [ ]:[KR].
- May 28, 2020: Added fixes for pepXML schema validation failures.
- April 28, 2020: Added the --brief-output option to tide-search, which prints only a subset of fields to the output file.
- April 27, 2020: Fixed build of Percolator to use BLAS libraries.
- March 18, 2020: Crux now builds MSTookit from original repository rather than a fork.
- December 16, 2019: Fixed a bug in Tailor method where minimal number of candidate peptides were selected incorrectly.
- October 18, 2019: Added res-ev p-value to performance-test. Fixed a bug in assign-confidence where columns were incorrectly shown when res-ev p-value was used as the score function.
- October 16, 2019: Fixed a bug in spectral-counts where the first amino acid of a peptide was be ignored if the flanking amino acids were unavailable.
- October 1, 2019: Added the "--static" option for percolator to enable the use of pretrained, static models.
- July 24, 2019: Fixed a bug in tide-search where delta_cn and delta_lcn were not correctly calculated for the following score functions: res-ev, res-ev p-value, and combined p-value.
- June 19, 2019: Fixed a couple of bugs in the calculation of residue-evidence and res-ev p-value. Correctly released memory when determining amino acid frequency. Added a bounds check when adding evidence to the residue-evidence matrix. Previously when calculating residue-evidence, all fragment ions were considered to be 1+ or 2+. Now fragment ions are only considered to be 2+ when the precursor charge is at least 2.
- April 16, 20219: Fixed Windows path parsing bug in spectral-counts.
- April 11, 2019: Changed the default parameters for tide-search so that the default mz bin width is 0.02 Da instead of 1.0005079 Da and the default precursor mass window is 50 ppm, rather than the previous default of 3 Da.
- November 6, 2018: Decoy generation will now allow duplicate decoy peptides within a decoy database and bewteen decoy databases.
- September 18, 2018: Modified the custom-enzyme flag to tide-index so that {X} can be used to indicate that all amino acids prevent cleavage. This is useful when providing a pre-digested set of peptides as input to tide-index.
- September 10, 2018: Fixed a bug in the calculation of the lnrSp feature in make-pin. Previously, the code took the log of the rank, indexed from zero. To avoid taking the log of zero, the code inserted the value zero when the rank was equal to zero. The effect was that both rank 0 and rank 1 received the same lnrSp score (i.e., zero). The fix is to do the calculation with rank indexed from one instead of zero.
- August 22, 2018: Fixed a bug causing filenames to be blank in Percolator outputs.
- August 14, 2018: Improved handling for terminal modifications when parsing peptide sequences.
- July 17, 2018: Added a hidden option called "use-old-atdc" to the assign-confidence command. This is included to allow comparison of the originally published version of aTDC with the new-and-improved version.
- July 16, 2018: Fixed a bug in how decoys are generated in tide-index. An off-by-one indexing problem led to some decoys being erroneously skipped even if they were not actually duplicates.
- June 19, 2018: Updated comet to release 2018012.
- May 30, 2018: Fixed a bug in spectral-counts that affected how the parsimony flag reported peptide ranks.

### Version 3.2.x

June 19, 2018

#### Major changes

- Crux on Linux now requires v 2.17 or higher of the C runtime libraries.
- Building Crux on Windows now requires Visual Studio 2017

#### Minor changes

- Fixed documentation errata
- Disabled network access by Comet
- Updated to Comet 2018012
- A 32-bit Linux version of Crux can be built on 64-bit Linux machines.
- Added support for parsing UNIMOD modifications.

### Version 3.2

May 20, 2018

#### Major changes

- Added the residue-evidence (res-ev), residue-evidence p-value (res-ev p-value), and combined p-value score functions to tide-index and cascade-search, and modified assign-confidence and percolator to handle these score functions. More details are available in "Combining high resolution and exact calibration to boost statistical power: A well-calibrated score function for high-resolution MS2 data".
- Implemented the "average target-decoy competition" protocol in `assign-confidence`. More details are available in "Progressive calibration and averaging for tandem mass spectrometry statistical confidence estimation: Why settle for a single decoy?".
- Added the localize-modification command to find the most likely locations of post-translational modifications on peptides from a set of peptide-spectrum matches.
- Improved handling of modifications in tide-index, allowing for indices containing many different types of modifications.

#### Minor changes

- Added 'equal-I-and-L' option to tide-index and spectral-counts commands. This directs the commands to treat I and L as equivalent amino acids.
- Percolator and make-pin can now accept multiple files on the command line.
- Updated Comet to version 2018.01 rev. 0.
- Implemented improvements to I/O speed for MzIdent format.
- Crux distributions are now identified using an abbreviated unique string from the version control system.
- Added support for mzML to qranker and barista.
- Modified tide-search to reports peak counts separately for each thread.
- Added "deisotope" option to tide-search.
- Added "max-charge-feature" option to make-pin so that the user can control how many charge features are created.
- Improved tide-index so that it will automatically recognize when too many temporary files will be required and to index modified peptides using an alternate method.
- Added the "mod-precision" option to control the number of digits used to represent post-translational modifications in various output files.
- Modified cascade-search so that the q-value threshold is not applied to the final database in the cascade.
- Tide-index will now clip methionines at the begining of proteins that do not have an internal clevage site.
- Enabled search-for-xlinks to search multiple spectra files, in any of the file formats supported by Proteowizard. Support for the `file-column` option added.
- Added the print-progress, concat, and mono-link options to search-for-xlinks and simplified the mod option.
- Changed the tide-search precursor-window option upper bound from 100 to 1,000,000,000 to allow open modification searching.
- Added PepXML support for mod_nterm_mass and mod_cterm_mass on modification_info.
- Removed the `feature-file-in` option to Percolator and added `top-match-in`, `train-best-positive` and `spectral-counting-fdr` options.
- Improved handling of the istopic-mass parameter in search-for-xlinks.
- Allow amino acids J (leucine or isoleucine), O (pyrrolysine, and U (selenocysteine) with the `tide-index` command.
- Added target/decoy column to tide-search and search-for-xlinks output files.
- Fixed mstoolkit to allow building with more recent versions of GCC.
- Fixed bug in tide-search that used total candidate count in output, rather than target candidate count.
- Fixed the percolator picked-protein option.
- Fixed error in handling of the custom-enzyme option.
- Fixed subtract-index so that it performs the subtraction only on the target peptides, and then removes for each subtracted target its corresponding decoy. Previously, the command did a simple subtraction separately on the targets and on the decoys.
- Fixed error that caused subtract-index to ignore the mass-precision option.
- Fixed error in search-for-xlinks that allowed too many missed cleavages.
- Fixed error in search-for-xlinks related to average mass.
- Fix an error in the processing of the peptide-list option to subtract-index.
- Fixed error in search-for-xlinks that attempted to use reversed decoys. search-for-xlinks only supports shuffled decoys.
- Numerous minor bug fixes and improvements to the documentation.
- Added 'How can I search my isotopically labeled data with Tide or Comet' to the FAQ.
- Added 'How do Tide and Comet handle combinations of static and variable modifications, as well as n-terminal modifications?' to the FAQ.
- Updated tutorial for test-fdr in percolator.
- Fixed a bug with target-decoy competition in assign-confidence when multiple matches had the same score.

### Version 3.1

January 10, 2017

#### Major changes

- Updated Crux to always use the latest version of Percolator on GitHub, rather than the most recent tagged release.
- Added the Param-medic command to perform automated inference of precursor and fragment m/z parameters. Param-medic options are also available for Comet and Tide.
- Added the isotope-error parameter to Tide.

#### Minor changes

- Added support for several new options to Percolator (search-input, tdc, subset-max-train).
- Allow assign-confidence to rank by Percolator score.
- Added +1 to the numerator of the FDR estimation when carrying out target-decoy competition. This correction was proposed by Barber and Candes, Annals of Statistics, 2015, as well as by He et al., arXiv:1501.00537, 2015.
- Fixed incorrect default value for the cpos parameter to percolator.
- Many other minor bug fixes and enhancements.

### Version 3.0

August 1, 2016

#### Major changes

- The `tide-search` command now supports threading. See the `num-threads` parameter for details.
- Added the `pipeline` command to run a series of commands, and the `cascade-search` to run searches across a series of databases.
- Renamed the command `calibrate-scores` to `assign-confidence` and completely revamped its functionality.
- The `search-for-xlinks` command offers parameters to control categories of candidates: `xlink-include-inter`, `xlink-include-intra`, and `xlink-include-inter-intra`.
- Added the `peptide-centric-search` option to tide-search.
- Compiling the Mac OS version now requires OS X Yosemite. It can be built from source using the Clang compilers distributed with the latest version of XCode.
- 32-bit and 64-bit Windows versions are now available, built using Visual Studio 2013.
- Updated Percolator to version 2.10.
- Updated Hardklor to version 2.30.
- Updated Comet to version 2016.01 rev. 1.

#### Minor changes

- The Windows version of Crux can be built with support for vendor specific file formats disabled.
- Added the `temp-dir` option to `tide-index`.
- The utilities `create-docs`, `subtract-index`, and `psm-convert` were added to Crux.
- The `generate-peptides` application has been revamped.
- Fixed a bug in `barista` that occurred when the number of peptides exceeded the number of PSMs.
- Precursor mass selection is fixed in `tide-search` when units are m/z.
- `hardklor` and `bullseye` now use standard logging.
- Version number added to log files. Additionally, the version numbers of `percolator`, `comet`, and `boost` are shown in the output of the `version` command.
- Short usage message is displayed on error (the full usage message is still displayed when a command is entered with no arguments).
- `feature-in-file` parameter added to `percolator`.
- The `seed` parameter for `percolator` has been renamed to `percolator-seed`.
- `comet` exits with return code 1 on failure, rather than 0.
- Default value for `use_sparse_matrix` changed to `1`.
- Default value for `use-neutral-loss-peaks` changed to `true`.
- `xlink-score-method` parameter added to `xlink-score-spectrum`.
- deltaLCn column added to `tide-search` tab-delimited output.
- Bug fixed in reporting flanking amino acids in `PostProcessProtein.cpp`.
- `Mix-max` procedure is updated to handle ties among target and decoy scores.
- Default value of `use-neutral-loss-peaks` changed to `true`
- `Exact p-value` calculation takes into account the flanking and neutral loss peaks.
- A bug was fixed in the naming of the Percolator input (.pin) file that is produced by Comet when the `output_percolatorfile` option is turned on. Previously, the file was named "comet.tsv"; now it is "comet.target.pin."
- `Assign-confidence` can take multiple input files.
- The `comet` command can take multiple input files.
- The `sidak` option has been added to `assign-confidence` to perform a Sidak adjustment.
- The `peptide-level` option has been added to `assign-confidence`.
- The refactored XCorr score has been re-scaled (by dividing by 20) to put it into a range that is comparable to that of the original XCorr score.
- The default value of `mz-bin-offset` has been changed from 0.68 to 0.40.
- The `percolator` command outputs the `pout` XML format again (controlled by the `pout-output` parameter).
- The `percolator` command can output its native output by using the `original-output` parameter.
- Fixed a bug in `tide-search` causing it to fail when `remove-precursor-peak` removed all peaks in a spectrum.
- The `max-precursor-charge` parameter for `tide-search` has been introduced.
- Fixed a bug in candidate peptide selection when m/z tolerance is used.
- Added the `read-tide-index` command, which reads an index produced by `tide-index` and prints a list of peptides it contains.
- Added the `stop-after` option to print-processed-spectra, which controls the point at which to stop preprocessing.
- The `tide-search` command now accepts multiple spectrum input files.
- Added the `use-z-line` option to specify whether precursor information is taken from the S or Z line when parsing MS2 files using ProteoWizard.
- Added missing options to the `percolator` command.
- The `tide-search` command may now accept a FASTA database in place of the index, in which case `tide-index` will be run prior to the search. The `store-index` option allows the generated index to be saved.
- The `assign-confidence` command will automatically detect score type if the `score` parameter is not specified, searching for xcorr, e-value, or exact p-values.
- The `cascade-search` will report the source index database of the peptide for all identified PSMs.
- The `top-match` parameter has been removed from `cascade-search`.
- `Assign-confidence` has got new options: "combine-modified-peptide" and "combine-charge-states".
- `Assign-confidence` now supports top-match>1 in peptide-level filtering mode.
- `Assign-confidence` can use smoothed-p-value scores for PSM ranking.
- `Assign-confidence` now does not carry out target-decoy competition in peptide-level filtering mode.
- Added the `xlink-assign-ions` and `xlink-score-spectrum` commands.
- Rearranged estimation methods in `assign-confidence`.
- Added top-match option for `estimation-method=peptide-level` case in `assign-confidence`.
- Modified assign-confidence so that ties during target-decoy competition are broken randomly.
- Changed the Percolator output feature "file" to contain the name of the file containing the spectrum (if available), rather than the name of the file containing the PSM.
- Added to `search-for-xlinks`, `xlink-assign-ions` and `xlink-score-spectrum` parameters of the form "use-a-ion" for a, b, c, x, y, and z-ions.
- Various other minor bug/leak fixes and performance enhancements.
- Added the `allow-dups` option to tide-index.

### Version 2.1

October 8, 2014

#### Major enhancements

- The `tide-search` command now supports calculation of exact p-values via dynamic programming, as described in this article. The p-value calculation is controlled with the `exact-p-value` parameter.
- `search-for-xlinks` now supports variable modifications and larger protein databases. This new code is disabled by default, but can be turned on by setting the parameter `use-old-xlink=F`.

#### Minor changes

- The default value of `mz_bin_offset` has been changed to 0.40 from 0.68.
- The `centroided` parameter for the `hardklor` command is now implemented.
- The `spectrum-format` parameter for the `bullseye` command works properly.
- The `tide-search` command now uses information from the Z line rather than the S line when reading spectrum files in the MS2 file format.
- The `tide-search` command now accepts the `spectrum-parser` parameter.
- The `protein-database` parameter to `spectral-counts` now only accepts fasta files. Parsing of a protein index is no longer supported.
- A bug was fixed in the `spectral-counts` command that prevented `tide-search` output files from being parsed correctly.
- The `default-direction` parameter now takes a string value of the feature name rather than an integer.
- The parameters `mz-bin-width` and `mz-bin-offset` have been added to `tide-search`.
- Percolator output now includes, for each PSM, the name of the file in which the spectrum resides.
- The parameters `clip-nterm-methionine`, `keep-terminal-aminos`, and `min-mods` have been added to `tide-index`.
- The parameters `use-flanking-peaks` and `use-neutral-loss-peaks` have been added to `tide-search`.
- The calculation of XCorr was simplified in Tide. In the new version, `tide-index` only generates peptides and sorts them, leaving the generation of theoretical peaks to be done entirely on the fly by `tide-search`.
- The `tide-search` command now prints search progress reports both to the screen and to the log file. The interval at which progress is printed can be controlled using the `print-search-progress` parameter.
- The `calibrate-scores` command now outputs files with the stem "calibrate-scores" rather than "qvalue."
- The `use-flanking-peaks` parameter now has a default value of false.
- The Comet search engine was updated to version 2014011.
- The MSToolkit parser was updated to the latest version (r73).
- The `version` command now outputs the revision number.
- Various improvements in performance and error handling were implemented.

### Version 2.0

June 6, 2014

#### Major enhancements

- Two new search engines are now included in Crux: Comet and Tide. The old search engine, search-for-matches, has been retired.
- Percolator has been updated to version 2.07.
- Crux now compiles in native Windows, rather than requiring Cygwin. Consequently, Crux running under Windows can parse vendor proprietary formats using the appropriate Proteowizard libraries.

#### Minor changes

- The "enyzme" parameters "lysc", "lysn", "arg_c", "glue_c", and "pepsin_a" were renamed to "lys-c", "lys-n", "arg-c", "glue-c", and "pepsin-a".
- Percolator outputs decoy files in addition to target files.
- Percolator tab-delimited peptides output now contain a posterior error probability (PEP) column.
- The Percolator tab-delimited peptide output has been corrected to show the PSM with the best score, rather than the worst.
- The Percolator tab-delimited PSMs output "matches/spectrum" value has been corrected.
- A new utility command, `make-pin`, is provided to create input files for use by Percolator.
- The `spectrum-min-mass` and `spectrum-max-mass` options have been renamed to `spectrum-min-mz` and `spectrum-max-mz`, respectively.
- A `spectrum-parser` option was added to `q-ranker` and `barista`.
- A bug was fixed in `q-ranker` and `barista` when reading peptides with modifications.
- Fixed the `q-ranker` tab delimited output so that, for a given peptide, flanking amino acids are reported for each protein that contains that peptide, rather than only reporting one set of flanking amino acids.
- A bug was fixed in `percolator`, wherein the first and last two characters were being truncated from peptide sequences when the input was missing the flanking amino acids or when the charge state was not indicated in the PSM IDs.
- The crux spectrum parser has been removed.
- Q-ranker now outputs decoy files.
- The Boost.Random library is now being used for random number generation.
- `calibrate-scores` no longer requires a protein input.
- `calibrate-scores` now only takes the top match per spectrum and charge.
- `get-ms2-spectrum` now prints z-lines with Bullseye files.
- Various performance and error handling improvements.

### Version 1.40

May 22, 2013

#### Major enchancements

- Crux Percolator was updated from version 1.05 to the latest release, version 2.04.

#### Minor changes

- The formula that is used to convert fragment m/z values from real numbers into integers was modified. Previously, the conversion was

        floor( (x / mz-bin-size) + 0.5 + mz-bin-offset )

  The new conversion formula is

        floor( (x / mz-bin-size) + 1.0 - mz-bin-offset )

  The default values of the two parameters (mz-bin-size and mz-bin-offset) have not changed. The allowed range of the mz-bin-offset parameter was previously [-1,1], but has been changed to [0,1].

  To understand the motivation for the changed formula, note that the mz-bin-offset parameter controls the location of bin edges, relative to integer masses on the mass scale. In general, mz-bin-offset should be chosen so that bin edges fall between the expected clusters of fragment masses. For fragments with 1+ charge, masses will cluster near integer values (after dividing by mz-bin-width), and the ideal value of mz-bin-offset would be 0.5. For mixtures of fragments with 1+ and 2+ charges, masses will cluster near integer and half-integer values, so mz-bin-offset near 0.25 or 0.75 would produce bin edges that best avoid the clusters of fragment masses. The old conversion formula did not correctly locate bin edges, given a chosen mz-bin-offset. The new conversion formula produces a proper translation of mz-bin-offset into bin edge locations.

- The `seed` parameter is now available to control the seeding of random number generator. The default value is 1. `seed` is available as a parameter from all applications. It can be set as a command line option for `crux search-for-matches` and `crux percolator`.
- The default value of the `decoys` parameter for `crux search-for-matches` was incorrectly documented. The default is actually `peptide-shuffle`, but the documentation said the default was `protein-shuffle`. The documentation has been corrected.
- The incorrectly labeled column header `xcorr rank` in barista and q-ranker feature files has been corrected to `xcorr score`.
- The q-ranker output file `qranker_output.xml` has been renamed to `qranker.xml`.
- The deltaCn computation has been modified to be consistent with Percolator. Rather than using

           deltaCni = (xcorr1 - xcorri+1) / xcorr1

  it is now computed using

           deltaCni = (xcorri - xcorri+1) / max(xcorri, 1.0)

- Percolator now accepts inputs in SQT, PepXML, and tab-delimited formats in addition to the PinXML format.
- Flags were added to several commands allowing the user to control whether various results files are created in the output directory:
  - `--sqt-output <T|F>` (search-for-matches)
  - `--mzid-output <T|F>` (search-for-matches, percolator)
  - `--pinxml-output <T|F>` (search-for-matches)
  - `--pepxml-output <T|F>` (search-for-matches, q-ranker, barista, percolator)
  - `--txt-output <T|F>` (search-for-matches, q-ranker, barista, percolator)
  By default, all of these flags are set to false except `txt-output`.

### Version 1.39

October 6, 2012

#### Major enchancements

- Crux is now released under an Apache license for all users.
- Parsing MS/MS spectra is now supported using Proteowizard (v. 3.0.3950). This allows Crux to handle MS/MS spectra in mzML (1.0 and 1.1), mzXML, MGF, MS2 and CMS2 formats. Use `spectrum-parser=pwiz` to enable Proteowizard parsing.
- Crux now uses `cmake` for the build process rather than `autoconf`/`automake`.

### Minor changes

- The Tide database search software is no longer distributed as part of Crux because its license is not compatible with the new Crux license.
- Support for PIN XML output was added to `crux search-for-matches`.
- Fixed a bug in `crux create-index` on Windows and Cygin that caused the program to fail with the message:

      WARNING: Cannot rename directory
      FATAL: Failed to create index

- The parameter `protein database` is now an option rather than a required argument for `crux spectral-counts`.
- mzIdentML file support has been added for `crux spectral-counts`.
- PepXML file support has been fixed for `crux spectral-counts` .
- Fixed bug in packedNorm() that sometimes generated incorrect values for the posterior error probability.
- Fixed bug in `crux create-index` on Windows and Cygwin that caused it to fail on Windows and Cygin.

### Version 1.38

July 20, 2012

#### Major enhancements

- Hardklor and Bullseye, tools for analyzing high-resolution precursor spectra, are now incorporated into Crux.
- Barista has been modified extensively so that it more closely resembles the other tools in Crux:

- Barista and Q-ranker accept search results in tab-delimited format, in addition to SQT format.
- Barista now reports posterior error probabilities, in addition to q-values, for PSMs, peptides and proteins.
- Barista's pep.xml output has been updated to be compatible with the TransProteomic Pipeline (TPP).

- The `sequest-search` command has been removed. The functionality of this command is still available using options for the `search-for-matches` command (see the frequently asked questions list for details).

#### Minor changes

- `crux predict-peptide-ions` calculates the mass shift resulting from the nh3 or h2o neutral loss correctly. Previously, a neutral loss resulted in an addition of mass rather than subtraction.
- `crux predict-peptide-ions` reports ion-type as 'b','y','a', or 'p' rather than a number (i.e. 0, 1, 2, 3).
- `crux predict-peptide-ions` now only allows one type of neutral loss modification per ion. Previously, multiple different types of neutral loss could be applied simultaneously to a single ion.
- The `neutral-losses` option was removed from `crux predict-peptide-ions`, due to its redundancy with the supported the `--nh3` and `--h2o` options.
- `crux predict-peptide-ions` supports 'bya' in the `--primary-ions` parameter, which will generate the a-ion series in addition to the 'b' and 'y' ions.
- The spectrum parsing is updated so that peaks with zero intensity are ignored.
- `crux spectral-counts` now has an option to compute the raw spectral count.
- `crux spectral-counts` now has an option to compute dNSAF values.
- A bug in `crux spectral-counts` was fixed so that it now properly normalizes the NSAF value.
- Multiple missed cleavages are now handled by `crux search-for-xlinks` using the missed-cleavages parameter. Previously, this was unsupported.
- Ambiguous amino acids are handled differently. 'J' is interpreted to indicate 'I' or 'L'. Peptides containing B, Z, and X are no longer be allowed, and a warning is issued when these amino acid codes are encountered.
- The stand-alone `crux-predict-peptide-ions` and `crux-generate-peptides` applications have been integrated into the main `crux` application.
- A bug has been fixed that affected the intensity adjustment of observed spectra when the bin width was set much larger or smaller than 1.
- Beginning with version 1.37, q-ranker erroneously included decoy PSMs along with target PSMs in the q-ranker.target.psms.txt file. All PSMs were sorted by q-ranker score so the targets and decoys were mixed together with no accompanying labels. This bug has been fixed so that Q-ranker once again returns only target PSMs.
- The search option `display-summed-masses` has been replaced by `mod-mass-format`. With this option, there is a new format for reporting modificiations: the mass in the square braces is that of the preceeding amino acid plus the modification mass. This format is compliant with TPP pep.xml output.
- Modifications are reported differently in the pep.xml header to be compliant with the TPP.
- The command `crux compute-q-values` now reports posterior error probabilities in addition to q-values. Consequently, the command was renamed `crux calibrate-scores`.
- Crux now provides an explicit error message when an incorrectly formatted MS2 file is parsed. Previously, crux simply reported that no spectra were found.
- Several additional scores were added to the `q-ranker` pep.xml files.
- Fixed the build procedure to work on both Lion and pre-Lion versions of OS X.
- Fixed a bug in the normalization of the observed spectra. Previously, after the 10-bin normalization of intensities, peaks with heights below 5% of the maximum were not being removed.
- When a fragmentation spectra file is read, the assumed peptide charge state information can be missing for the scans. In cases where this information has not been provided, Crux will now estimate the possible charge states.
- The feature files for Q-ranker and Barista now contain a header describing each feature.
- Q-ranker and Barista now output all of the fields provided in the input search files.

### Version 1.37

December 22, 2011

#### Major enhancements

- Added `crux barista`, a tool for inferring protein identifications.
- Reimplemented `crux q-ranker` with new outputs and command-line syntax.
- Percolator and compute-q-values now compute and report posterior error probabilities, in addition to q-values.
- `crux create-index` now generates and store decoy peptides in the index. Decoys can still be generated on-the-fly when searching .fasta files.

#### Minor changes

- A pair of options, `cterm-fixed` and `nterm-fixed`, allow fixed terminal peptide modifications, i.e., a mass shift applied to every peptide on either or both termini.
- A bug in the spectrum processing code was fixed, which was erroneously eliminating a few of the highest m/z fragment peaks. Xcorrs have changed slightly as a result.
- Simplified the `--mz-bin-width` and --mz-offset parameters so that `--xcorr-var-bin` is no longer needed.
- Indexes now store information about any static mods used when they were created.
- In the tab-delimited output files the 'matches/spectrum' column gives the number of target peptides compared to the spectrum and the decoy file has an additional column, 'decoy matches/spectrum' that gives the number of decoy peptides compared to the spectrum. These two numbers may differ when decoys are not generated on-the-fly.
- The header lines of Barista and QRanker include all of the columns in the search results.

### Version 1.36

August 4, 2011

#### Tide changes

- Added support for user-specifiable variable and static amino acid modifications.
- Tide's version of XCorr has been modified so that it is always identical to those of a recent version SEQUEST®.
- Added support for user-specifiable maximum number of missed enzyme cleavage points.
- Tide now performs basic charge state inference when this information is missing from the spectrum input files.
- User-specifiable output formatting, using new the `tide-results` utility, including
  - Text format with user-specifiable fields.
  - SQT format.
  - PepXML format.
  - Choose whether to display all proteins in which each peptide occurs or just one representative.
- Note that new file formats are created by `tide-index`. Index files created by previous versions of Tide should not be used with the new version.

#### Minor changes

- Q-ranker now reports q-values instead of false discovery rates.
- Corrected several errors in the pep.xml output, related to reporting the spectrum filename, charge, and neutral mass.
- Changed the `missed-cleavages` option to allow specifying the maximum number of missed cleavages in a peptide rather than specifying none/any.
- Fixed a bug that did not print the spectrum file name correctly in the pep.xml files.
- Improved the error message produced by `crux spectral-counts` when the input PSM file does not contain the required q-values.
- Previously, Crux's theoretical spectrum includes two flanking peaks around each b- and y-ion. The new Boolean flag `use-flanking-peaks`, allows these to be either on or off. Flanking peaks are by default in `crux sequest-search` but are not used by default in `crux search-for-matches`.

### Version 1.35

March 23, 2011

#### Major enhancements

- Added the `crux spectral-counts` command for estimating protein quantification.

#### Minor changes

- The spectrum neutral mass is now calculated from the m+h value provided by the Z-lines of an MS2 file. Also, multiple z-lines of a spectrum that have the same charge are now supported. With these changes, `crux search-for-matches` and `crux sequest-search` can now take advantage of accurate precursor masses in MS2 files generated by Bullseye (Hsieh et al. J. Proteome Res. 9(2):1138-43, 2010).
- Made the `pep.xml` file header produced by Crux compatible with the Transproteomic Pipeline.
- Introduced several changes to improve performance on MacOS X.
- Added an `nterm` option to the link argument of `crux search-for-xlinks`.

### Version 1.34

December 22, 2010

#### Minor changes

- The default value of the "pi-zero" parameter has been changed from 0.9 to 1.0.
- A new option `--peptide-list` has been added to `crux create-index`. When set to T, this option causes an ASCII file of peptides to be included in the output directory. Each line of the file lists the peptide sequence and its neutral mass.
- The default bin offset was returned to 0.68. It had erroneously been changed to 0 in version 1.33.
- A new option `--compute-sp` has been added to `crux search-for-matches`. When set to true, all candidate peptides will be scored by Sp in addition to xcorr. This option is recommended for generating input to `percolator` or `q-ranker`.
- Added `max-ion-charge` parameter to designate the maximum charge for theoretical ions in `crux search-for-xlinks` and `crux xlink-assign-ions`.
- Modifications may optionally indicate if they prevent cleavage or prevent cross linking.
- Results are printed to .pep.xml files in addition to .txt files.
- Added a `mass-precision` option which sets how many digits are written for all mass and m/z values in .txt, .sqt, and .ms2 files.
- The columns printed to the .txt files now vary with the `crux` command being run and the settings so that unused columns are not printed.
- Fixed a bug that limited what files were used as input for post-search commands (e.g. `crux percolator`) based on fileroot. Now files with different fileroots can be analyzed together.
- Fixed a bug that caused searches using fasta files to search peptides multiple times if they appeared in a file multiple times.

### Version 1.33

July 7, 2010

#### Major enhancements

- The command line syntax for `compute-q-values`, `percolator` and `q-ranker` was modified to allow the programs to read from and write to separate directories.
- A bug in q-ranker and percolator was fixed. This bug was introduced several versions ago, and it led to erroneously many PSMs receiving very small q-values. The problem relates to the scaling of the delta Cn feature, and the fix is a temporary stopgap: we remove the feature entirely. The next version should have the corrected feature in place.

#### Minor changes

- A new option `--min-peaks` has been added to set a filter for the minimum number of peaks a spectrum must have in order for it to be searched. The default minimum is 20 peaks.
- The hardcoded limit for the number of proteins allowed in a database has been removed.
- The hardcoded limit for the number of spectra and the number of peaks/spectrum has been removed.
- A new option `--xcorr-var-bin` has been added to toggle the new binning of the m/z axis. The default bin offset was returned back to 0, but the default bin width is still 1.000508. (Note: this erroneous change was subsequently reverted in version 1.34.)
- The option `--max-ion-charge` now places a limit on the maximum charge state of the ions generated for the xcorr theoretical spectra for `crux search-for-xlinks`. This change is also supported by `xlink-assign-ions`.
- The option `--no-xval` was added to q-ranker, allowing faster execution by skipping the internal cross-validation to select hyperparameters.
- The precision of modification masses written to the .txt files now matches the maximum precision given in the parameter file.
- Sub-options `prevents cleavage` and `prevents cross-link` have been added to variable modifications and support has been provided for the `"prevents cleavage"` sub-option.

### Version 1.32

July 6, 2010

#### Major enhancement

- Version 1.32 includes changes only to Tide. A new binary is introduced — a modified version of ProteoWizard's msconvert program — that is capable of converting spectrum data from a wide range of file formats into Tide-readable .spectrumrecords input files.

#### Minor changes

- tide-import-spectra is removed from the distribution. Although it was faster than msconvert, it was specific to ms2 files, whereas msconvert now works with any ProteoWizard-supported file format.
- A standalone utility for reading binary .spectrumrecords files is introduced, called read-spectrumrecords. This program is useful, for example, for visually checking the output of msconvert.
- Demo input files worm-06-10000.spectrumrecords and yeast-02-10000.spectrumrecords now replace their counterparts worm-06-10000.ms2 and yeast-02-10000.ms2. With the introduction of Tide-compatible msconvert, ms2 files are in no way special to Tide anymore. Any ProteoWizard-supported file format can be converted to the .spectrumrecords format for use with Tide. Starting with data files in Tide's spectrumrecords format eliminates the conversion step in the Tide search demo.
- The old demo scripts worm-demo.sh and yeast-demo.sh have each been replaced by a pair of new scripts that demonstrate the indexing and searching steps separately. This is in order to highlight the two-step process and the fact that indexing needs to be done just once for each fasta file, whereafter the index may be reused indefinitely to perform searches.

### Version 1.31

June 2, 2010

#### Major enhancements

- This version of Crux is released with a demo version of a new search engine, Tide. Tide is an independent reimplementation of the SEQUEST® algorithm. The immediate ancestor of Tide is the Crux `search-for-matches` command, but Tide has been completely re-engineered to achieve a thousandfold improvement in speed while exactly replicating Crux XCorr scores. Currently, Tide is not fully integrated with Crux, and is available only in binary executable format.

#### Minor changes

- Two options have been added to `crux search-for-matches` (`--mz-bin-width` and `--mz-bin-offset`) to allow control of the binning of the m/z axis.
- The `ion-tolerance` option was removed from the search tools.
- The default location of the left edge of the first bin along the m/z axis is 0.68, rather than 0.0. This change makes bin edges less likely to fall near fragment peak locations. The default bin width has also changed from 1.001141 to 1.000508
- The log file now includes information about the date and time that the command was issued, the name of the computer on which the command was run, and the elapsed wall clock time at the end of the run.
- The command line option `--version T` has been replaced with a command `crux version` that prints the version number to standard output and then exits.
- The header for the feature file produced optionally by `crux percolator` and `crux q-ranker` now includes names for the first two columns, scan number and label (i.e. target or decoy peptide).

### Version 1.30

May 5, 2010

#### Major enhancements

- A new command, `xlink-search`, has been added. This command searches a collection of spectra against a sequence database, finding cross-linked peptide matches. The algorithm was described in the following article:

> Sean McIlwain, Paul Draghicescu, Pragya Singh, David R. Goodlett and William Stafford Noble. "Detecting cross-linked peptides by searching against a database of cross-linked peptide pairs." Journal of Proteome Research. 2010.

- A new search command, `sequest-search`, was added. The original command, `search-for-matches`, behaves as before except that no .sqt files are printed and no Sp scoring is performed. The new `sequest-search` emulates SEQUEST® searching. It first scores all candidate peptides with the Sp score, then ranks and filters the results by that score, scoring the remaining candidates with xcorr. Results are printed to .txt and .sqt files.
- The .mzXML file format is now supported for crux search-for-matches and crux sequest-search when the `--use-mstoolkit` option is set to TRUE.
- The .csm output files from `search-for-matches` are no longer produced and post-search operations take .txt files as input.

#### Minor changes

- Replaced `mass-window` parameter with `precursor-window` and added `precursor-window-type` for selecting windows of type mass, m/z, or ppm.
- The `feature-file` option is now true/false instead of taking a file name. The file is named '<fileroot>.<qranker| percolator>.features.txt'. A header with the column names was added to the file.
- The new option, `--scan-number`, performs a search on a specified subset of the spectra in the given file.
- When only one decoy file is produced, the name is now 'decoy.txt' instead of 'decoy-1.txt'
- In the .txt files, protein names are now followed by the start index of the peptide.
- Modified sequences are reported differently in the .txt files. Instead of modifications being represented by symbols (*,@,#, etc.) they are indicated with the mass shift of the modification within square brackets. As before, the modification information follows the residue that is modified. If multiple modifications appear on one residue, the masses may either be summed together or printed separately in a comma separated list. This behavior is controlled by the `--display-summed-mod-masses` option.
- The modified decoy sequences are generated differently. Before, all modified peptides were generated and each one was shuffled to create a decoy peptide. Now a peptide is shuffled once for each peptide_mod and all modifications are applied to that same shuffled peptide.
- The 'percolator rank' column is now based on percolator scores instead of percolator q-values. For cases where two PSMs have different percolator scores but the same q-value, the ranks will reflect the score differences.
- The Weibull parameters used for computing p-values are now printed to the .txt files.
- The reporting of DeltaCn has changed. For PSMs of rank i, deltaCn used to computed be as deltaCn_i = (xcorr_0 - xcorr_i) / xcorr_0. Thus, deltaCn for the top-ranked PSM was always 0. Now deltaCn_i = (xcorr_0 - xcorr_i+1) / xcorr_0. This change only affects `search-for-matches`, not `sequest-search`. It also means that `q-ranker` and `percolator` use the correct DeltaCn values.
- A new command, `print-processed-spectra`, performs XCorr-style pre-processing on all spectra in a given file.
- A small bug was fixed, in which even if the user requested average mass, the search programs still used monoisotopic mass in the calculation of XCorr and Sp.
- Protein fasta files are now parsed protein-by-protein so that parsing large files does not use excessive amounts of memory.
- A limit was removed on how many peptides a protein can produce.
- Crux is now built with g++ instead of gcc.

### Version 1.22

September 16, 2009

#### Major enhancements

- Crux is now distributed in two versions, a full version that is covered by the same type of license as before (free to non-profit users, and via a licensing fee to commercial users), as well as a stripped-down version that is released under an open source license. The stripped-down version does not include the database search functionality but does include all of the post-processing tools. We are unable to release the entire Crux package under an open source license due to intellectual property issues. Both versions of Crux are available via the Crux web page: http://noble.gs.washington.edu/proj/crux/.
- A new tool, q-ranker, is available for estimating peptide-spectrum match q-values. This tool was described in the following article:

  > Marina Spivak, Jason Weston, Leon Bottou, Lukas Käll and William Stafford Noble. "Improvements to the Percolator algorithm for peptide identification from shotgun proteomics data sets." Journal of Proteome Research.

- Version 1.05 of `percolator` has now been integrated into the Crux source tree. A separate installation of `percolator` is no longer needed for basic `percolator` functionality. Note, however, that `percolator` remains under active development. You may therefore wish to install the current, stand-alone version of `percolator` and run it separately to take advantage of new features.

#### Minor changes

- The internal normalization of the observed spectra has been modifed to drop those peaks whose intensity is less than 1/20 of the maximum intensity in the spectrum. This brings the xcorr score for `crux` into closer agreement with the xcorr score for SEQUEST®.
- Compute-q-values now generates three different q-values (1) from p-values using an analytical null model, (2) from decoys and xcorr using an empirical null model, or (3) from decoys and p-values using an empirical null model. All three types of q-values are computed when p-values and decoys are present in the search results.
- A parameter file is now automatically written to the output directory.
- A log file recording messages sent to stderr has been added for `search-for-matches`, `compute-q-values`, and `percolator`.
- The `--use-mz-window` parameter is now available for `search-for-matches`. When enabled, peptides must be within +/- 'm/z-window' of the spectrum m/z. The m/z-window value is taken from `mass-window`.
- A numerical bug in the Weibull p-value calculation was fixed, which had previously caused occasional erroneous NaNs to be output.
- The Weibull estimated p-values generated by `search-for-matches` are now returned as p-values instead of as -log(p-value). The corresponding q-values returned from `compute-q-values` are also now returned without the -log transform.
- The `--precision` option has been changed to control the total number of significant digits printed instead of the number of digits after the decimal point. The default precision has changed from 6 to 8.
- The parameters estimated for the Weibull distribution (used for computing p-values) now use the xcorrs from all PSMs for a spectrum instead of a random selection of 500.
- The estimation of Weibull distribution parameters requires a minimum number of scored PSMs. In the previous version, spectra with fewer PSMs than the minimum were not given a p-value. Crux will now generate extra decoys until there are enough scores.
- The p-values for decoy PSMs are now generated from the same Weibull distribution parameters as are used for the targets of the same spectrum.

### Version 1.21

May 14, 2009

- The output for `search-for-matches`, `compute-q-values`, and `percolator` has been revised extensively. `crux` will now create a directory, and all output files will be created in that directory. By default the directory will be named `crux-output`, but this can be changed using the new `output-dir` option.
  The output files for `search-for-matches` will be:
  - `search.target.csm`
  - `search.decoy-?.csm`
  - `search.target.sqt`
  - `search.target.txt`
  The output files for `compute-q-values` will be:
  - `qvalues.target.sqt`
  - `qvalues.target.txt`
  The output files for `percolator` will be:
  - `percolator.target.sqt`
  - `percolator.target.txt`
- The `fileroot` option has been added. This option is used to specify a string which will be added as a prefix to all output files.
- The option `cleavages` was replaced with two options, `enzyme` which specifies the name of an enzyme (e.g.trypsin) and `digestion` which indicates the degree of specificity, partial or full digest. The full list of available enzymes is in the html docs and in the usage statement. See also `custom-enzyme` below.
- The option `custom-enzyme` allows users to define arbitrary digestion rules. This overrides the `enzyme` option. Syntax for the custom digestion rule is the same the syntax used by X!Tandem and is described in the html docs.
- The number of PSMs per spectrum printed to the output files is now controlled by one option, `top-match`. This makes `max-sqt-result` obsolete.
- It is now possible to control how many decoy sequences are generated and in which file(s) they are returned. There is a new option, `num-decoys-per-target`, which can be used to generate more than one shuffled peptide per spectrum. This replaces `number-decoy-set`.
- A new option, `decoy-location` has been introduced. The three possible values are 'target-file' where all PSMs (target and decoys) are sorted together for each spectrum and returned in one file, 'one-decoy-file' where target PSMs are printed to one file and all decoys are printed to another, and 'separate-decoy-files' where there are as many decoy files as there are decoys per target.
- Protein names for decoy matches are now prepended with 'rand_' in the SQT files as in 'L rand_Y45678'.
- The option `unique-peptides` only applies to `crux-generate-matches`. Each peptide is stored in the index exactly once with references to all protein sources. Searches with fasta files print each peptide only once.
- The precision of the masses and scores printed to the sqt and text files can now be specified by the user. The default precision changed from 2 to 6.
- Search progress is now reported by printing every 10th spectrum that is searched. The verbosity can be adjusted with the parameter `print-search-progress`.
- Decoy (shuffled) sequences now keep the first and last residue the same as the target sequence that was shuffled to produce it. This is a reversion to previous behavior.
- It is now possible to skip the Sp score and score all PSMs with xcorr. The default procedure is still to score all peptides for one spectrum with Sp, rank by Sp, and eliminate all but the best-ranking PSMs (by default, the top 500). The remaining PSMs are scored by xcorr, re-ranked by xcorr and the top results returned. By setting max-rank-preliminary=0, the Sp scoring is skipped and xcorr is computed for all PSMs.
- A new parameter `reverse-sequence` can be used to generate decoy peptides by reversing them rather than shuffling. The first and last residues are left unmoved. If the sequence is a palindrome , then a decoy will be generated by shuffling and a note to that effect will be printed at the DETAILED INFO level of output (verbosity = 40).
- P-values are now computed for decoy peptides.
- The algorithm used to calculated the xcorr score has been modified so that xcorr score will be in better agreement with scores generated by SEQUEST®.

### Version 1.20

January 6, 2009

- Generating peptides and searching with up to eleven different dynamic modifications is now possible. New options associated with this feature are mod, cmod, nmod, max-mods, max-aas-modified.
- The format of the .csm files has changed and files written by older versions of crux are not readable with crux version 1.2.
- When the option `cleavages` is set to `all`, peptide generation ignores all tryptic cleavage sites, effectively setting the `missed-cleavages` option to `TRUE` regardless of user settings.
- When one spectrum has identical xcorr scores for different sequences, the rank of all those matches will be the same. Matches with the next highest score will rank one below.
- The options for setting the preliminary and primary score type have been removed and are fixed as Sp and xcorr, respectively. A new option, `compute-p-values=<T | F>`, was added to control p-value computation.
- The SQT file contains the spectrum calculated mass instead of observed mass/charge on the S line.
- There is now a test for confirming that the file downloaded from the crux website was not corrupted. See installation instructions for details.
- Calculating a p-value requires a minimum of 40 matches. Spectra with fewer than 40 matches will have p-value scores returned as NaN and a warning will be printed at the DETAILED_INFO level (40) of verbosity.
- Fixed error in generating neutral-loss peaks created as part of the theoretical spectrum.

### Version 1.02

December 1, 2008

- Three programs, `crux-create-index`, `crux-search-for-matches`, and `crux-analyze-matches`, were merged into one program named `crux`.
- Percolator is now truly optional as all Crux programs will build without it.
- Fragment masses can now be calculated as average or mono-isotopic. This is controlled by the `fragment-mass` option in the parameter file.
- The name of the score-type option that calculates p-values was changed from xcorr-logp to xcorr-pvalue.
- SQT files have two new lines in the header which describe the arrangement of values in the results.
- HTML documentation was updated to reflect the above changes.

### Version 1.01

October 15, 2008

- A bug limiting the length of the name of an index file was fixed.
- Modifications were made so that Crux will build with version 1.05 of Percolator. This is the only supported version of Percolator.
- Memory leaks in `crux-search-for-matches` were patched.
- The `--version` option was added.

### Version 1.0

March 4, 2008

Initial release