HiTIME is a program for identifying twin-ion signals in Liquid chromatography-mass spectrometry (LCMS) data. HiTIME is designed to be integrated with existing analysis pipelines, such as those created with OpenMS.
HiTIME filters twin-ion signals in LCMS data. This process re-weights each data point to a Z-score of how well the point matches an idealised twin-ion signal versus alternative ion signatures.
- The
hitimeprogram takes an mzML file as input and produces an mzML file as output. Intentsity values in the input file which correspond to the lower mass in a twin-ion peak are retained and scored highly in the output, all other intensities are downweighted towards zero.
HiTIME is released as open source software under the terms of the 3-Clause BSD License. See the contents of the file called LICENSE in the top level of the source code repository for a copy of the terms.
HiTIME is provided as a Docker container, which can be installed like so (assuming you have docker installed on your computer):
docker pull bjpop/hitime
Docker can be installed on all modern operating systems. Please review the Docker installation instructions for more information.
HiTIME can be build from source by following the instructions in the notes folder:
- Mac OS X:
notes/build.osx.sh - Linux:
notes/build.linux.sh
Run this command in the top-level directory of the source tree:
docker build -t bjpop/hitime .
We provide a convenient wrapper script to run the HiTIME docker container.
Test data is included in the data folder within the repository. Running the following command
should produce meaningful output saved in results.mzML.
./hitime-docker.sh -i data/testing.mzML -o results.mzML -- -d 6.0201 -r 10 -m 230
Note that the wrapper script accepts arguments in a slightly different format than the regular executable program: the -i and -o arguments first, then all other arguments are specified after a -- flag.
You might see some warnings in the output which complain about the format the of input testing.mzML file. You can
safely ignore them. It is just OpenMS being strict about the format of the file.
-h, --help Show this help information.
-l, --mzlower arg Lower M/Z offset for local max window, e.g. 0.3.
Must be used with 'mzupper', can not be used with
'mzwidth'
-u, --mzupper arg Upper M/Z offset for local max window, e.g. 0.7. Must
be used with 'mzlower', can not be used with
'mzwidth'
-a, --iratio arg Ratio of doublet intensities (isotope / parent).
Defaults to 1.000000
-r, --rtwidth arg Full width at half maximum for retention time in
number of scans. Eg: 10
-m, --mzwidth arg M/Z full width at half maximum in parts per million.
Eg: 230. Must be used with 'mzdelta', can not be used
with 'mzlower' and 'mzupper'.
-d, --mzdelta arg M/Z delta for doublets. Eg: 6.0201. Must be used with
'rtwidth' and 'mzwidth', can not be used with
'mzlower' and 'mzupper'.
-z, --confidence arg Lower confidence interval to apply during scoring (In
standard deviations, e.g. 1.96 for a 95% CI).
Default: ignore confidence intervals
--debug Generate debugging output
--version Print version number and exit
-j, --threads arg Number of threads to use. Defaults to 1
-c, --cache arg Number of input spectra to retain in cache. Defaults
to 50
-i, --infile arg Input mzML file
-o, --outfile arg Output mzML file
Note that the examples below illustrate how to call the executable program directly. If you wish to use the Docker wrapper scripthitime-docker.sh then you will need to adjust the syntax accordingly; see the notes above about using the wrapper script.
hitime -j 4 -i data/testing.mzML -o results.mzML -d 6.0201 -r 10 -m 230
for a computation using 4 threads, where data/testing.mzML contains the input mass spectrometry data in mzML format, and the output file is called results.mzML.
The parameters defining the taget twin-ion signal are, -d 6.0201 the M/Z diference between the natural and heavy isotope versions of the precursor, -r 10 the retention time (RT) full width half maximum (FWHM) size in number of RT steps (scans), -m 230 the M/Z FWHM size in parts per million (ppm). These values can be determined by measurement of the precursor signal in standard visulisation software.
HITIME can also be used to filter the data to only output the data point that has the largest value in a region defined by the Retention Time (RT) full width half maximum (FWHM) size, and the M/Z upper and lower bounds. E.g.:
hitime -i results.mzML -o max.results.mzML -r 10 -l 0.3 -u 0.7
This will produce two files, max.results.mzML and max.results.csv. The CSV file is a comma separated text file listing the local maxima. This list can be sorted to help identify the strongest twin-ion signal matches. The fields are RT, M/Z, score.
HITIME assumes that the input mzML file is indexed. To index an input file, the OpenMS FileConverter tool can be used, eg:
FileConverter -ini openms.ini -in input.mzML -out input_indexed.mzML
An example openms.ini can be found in notes/openms.ini.
curl -sL https://melbourne.figshare.com/ndownloader/files/14131079 > BSA_SUBSET.mzML
A pure protein that was reduced, reacted with heavy/light paracetamol and then digested. It contains lots of twin ions. Because these twin-ions are mostly peptide derivatives, they come in multiple charge states which effectively gives us a few different doublet spacings. There are many separated by 6.0201, a good number separated by 3.01005 and a few separated by 2.0067. We have removed any points with intensity <1000 to make the file smaller.
curl -sL https://melbourne.figshare.com/ndownloader/files/14131085 > BSA_FULL.mzML
A control dataset to match the larger dataset above, that contains no true twin ions.
curl -sL https://melbourne.figshare.com/ndownloader/files/16249331 > BSA_FULL_CONTROL.mzML
Synthetic LC-MS data was produced that contain exactly 17 twin-ions of varying abundance in addition to a number of non-twin-ion signals.
curl -sL https://melbourne.figshare.com/ndownloader/files/16247657 > synthetic17.mzML