The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data
© Pascal et al; licensee BioMed Central Ltd. 2007
Received: 11 January 2007
Accepted: 16 May 2007
Published: 16 May 2007
The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange) experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues.
We have developed an integrated software system for the automated analysis and representation of H/D exchange data that has been titled "The Deuterator". Novel approaches have been implemented that enable high throughput analysis, automated determination of deuterium incorporation, and deconvolution of overlapping peptides. This has been achieved by using methods involving iterative theoretical envelope fitting, and consideration of peak data within expected m/z ranges. Existing common file formats have been leveraged to allow compatibility with the output from the myriad of MS instrument platforms and peptide sequence database search engines.
A web-based interface is used to integrate the components of The Deuterator that are able to analyze and present mass spectral data from instruments with varying resolving powers. The results, if necessary, can then be confirmed, adjusted, re-calculated and saved. Additional tools synchronize the curated calculation parameters with replicate time points, increasing throughput. Saved results can then be used to plot deuterium buildup curves and 3D structural overlays. The system has been used successfully in a production environment for over one year and is freely available as a web tool at the project home page http://deuterator.florida.scripps.edu.
The automated calculation and presentation of H/D exchange data in a user interface enables scientists to organize and analyze data efficiently. Integration of the different components of The Deuterator coupled with the flexibility of common data file formats allow this system to be accessible to the broadening H/D exchange community.
Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry (MS) has proven to be a powerful technique for the study of protein structure and dynamics as well as protein-protein and protein-ligand interactions [1, 2]. The rate at which an amide hydrogen exchanges with solvent deuterium reveals much about its local environment. Changes in protein structure or dynamics can result in changes in H/D exchange kinetics for a subset of amide hydrogens along the protein backbone. Measurement of these alterations in kinetics provides chemical sensors for changes in protein structure and dynamics.
To perform these experiments the target protein or protein complex of interest is subjected to a time course of incubation with deuterium containing buffer to initiate on-exchange of deuterium into the protein. At prescribed time intervals, aliquots of the on-exchange mixture are rapidly quenched by the addition of a low pH and low temperature buffer containing denaturants to prevent further on-exchange, minimize off-exchange of incorporated deuterium and help denature the protein. The quenched and denatured protein is then subjected to enzymatic digestion by acid stable proteases. The resultant peptides are collected on a reverse-phase HPLC column and then gradient eluted directly into a mass spectrometer equipped with an electrospray ionization source where the mass of each peptide is measured. Alternatively, peptides may be spotted onto a target and analyzed with matrix assisted laser desorption ionization (MALDI) mass spectrometry [3, 4]. By repeating this process for all on-exchange time points the mass increase of each proteolytic peptide over the time course of deuterium exposure can be determined. From these data the rates of deuterium exchange can be determined for the corresponding areas of the intact protein.
Since the inception of H/D exchange mass spectrometry, substantial advancements in methodology, automation and instrumentation have been made, [5–7] however, processing, curation, and validation of the large number of data points produced in a typical H/D exchange experiment [2, 8] presents a significant data processing and analysis challenge. These issues have in part been addressed by several existing software tools and algorithms. Interactive software to simplify the calculation of average molecular weight has been developed . Other algorithms are implemented as command line software which automate the extraction of deuterium content with Fourier transform and natural isotopic abundance based calculations [10, 11]. Proprietary software has also been described that automates the extraction of deuterium content in a standalone software application [7, 8]. Peak width analysis approaches have been created  as well as Excel tools leveraging this approach . Deconvolution techniques have been developed to determine deuterium content in less straightforward situations [14–16]. These above mentioned approaches have shown success in given cases, and their efforts serve to illustrate the interest and challenge of this area, however, the processing and management of H/D exchange data remains a significant bottleneck in the technique.
While progress has been made in H/D exchange data processing, these solutions have not always been easy to employ, and thus have not been widely adopted. Some solutions are command line based, and others are not freely available or their methods have not been described in detail. Often users are required to run separate programs, covering different functions such as peptide location and centroiding. In addition, output from the wide variety of MS instruments and database search engines presents another hurdle in the creation of broadly applicable solutions. There are no published H/D exchange software solutions based on platform independent common mass spectrometry data file formats, such as mzXML. The large size and number of files in conventional studies overburdens users with organizing, migrating and storing data, a critical factor that has yet to be addressed. Integrated user interfaces are also required that allow for the presentation, curation and validation of the results. These factors illustrate the need for software that is precise, efficient, convenient to use, and freely available to the scientific community.
A web-based software system called "The Deuterator" is presented here. It is freely available as an online service, and addresses many of the needs described above. The software can be used to precisely determine the deuterium content for protein segments in an integrated, high-throughput manner, taking steps toward instrument platform independence through the use of common data file formats. Novel tools have been implemented and are accessible through a user interface providing functionality such as the determination of percentage deuterium incorporation for a given peptide. The software is compatible with data from most common database search engines and is compatible with high and low-resolution mass spectral data. Although the software was developed for analyzing LC ESI-MS data, processing data from H/D experiments that do not incorporate a chromatographic step or data that were generated with MALDI MS instruments are supported equally. The output for each peptide included in the experiment is an accurate measurement of deuterium incorporation over time, which can then be plotted as deuterium vs. Log time graphs. Peptide sequences can be positioned next to the protein sequence to display the sequence coverage. Graphical representations of H/D exchange data can then be added by using two-dimensional color maps to represent the data values, which may also be overlaid onto a high-resolution 3D protein structure. To develop and validate this web-based tool, we tested the system in a production environment for over one year with both low (~2000) and high (>60,000) resolution ESI-MS data.
The Deuterator is available as a web-based application. The back-end architecture was built with Java and the online service was implemented with JSP. The MS data viewers were implemented using open source software [17, 18]. Mass spectral and search result data are required in native format, and are converted into common file formats. Protein sequence data is required in FASTA format.
The broad range of mass spectrometers and sequence search tools has resulted in a large number of differing file formats, resulting in compatibility problems for many new and existing software packages. This issue has been addressed by recent efforts to create the common file formats mzXML  and mzData [20, 21] for mass spectrometric data. One such effort  has developed the common formats mzXML and pepXML for mass spectral data and peptide identities respectively, and provides freely available mzXML conversion programs for various instrument formats. The Deuterator leverages these files formats and therefore takes steps toward greater compatibility with a broad range of mass spectrometers and protein search engines.
The components of The Deuterator are best viewed in a browser with tabbed browsing functionality, allowing for many time points to be viewed simultaneously. Results from the calculations are saved in a flat file format (CSV), which are easily viewed with applications such as Microsoft Excel.
Results and discussion
There are three user inputs into The Deuterator: 1) The mass spectrometry data files (mzXML format); 2) The protein sequence file in FASTA format; and 3) A list of the peptides to be included in the H/D exchange experiment that we refer to as the "peptide set".
Accounting for the charge state of the peptide, the 0% deuterium incorporation minimum, mz0, is defined as being the mass to charge ratio (m/z) value for the peptide's monoisotopic m/z. The 100% deuterium incorporation m/z limit, mz100, for the given protein segment is then calculated according to equation where n = number of amino acids in the peptide, p = the number of non-N-terminal prolines, and z = charge.
mz100 = mz0 + ((n - p - 2)/z) (1)
The amino acid proline has no amide hydrogen and so they are subtracted from the equation. The subtraction of 2 in the equation is explained elsewhere . The values of mz0 and mz100 are used in subsequent calculations and are presented in the interface to serve as positional markers. These values are different from MZ Start and MZ End , which are the user-defined limits of the mz range, used to define the centroid calculation.
Deuterium exchange calculations
The calculation of the amount of backbone deuterium exchange can be accomplished using one of two methods, the "centroid" method or the "theoretical fit method".
A different approach is taken for the "centroid" method. Variables m/z minimum and m/z maximum are initially assigned to mz0 -1 Da and mz100 +1 Da, and are adjustable. XY data between m/z minimum and m/z maximum are used to calculate the intensity weighted mean m/z centroid value. The final centroid result is then used to calculate the percentage of deuterium incorporation with methods outlined previously .
where f0 = mz0, z = charge state, i is an integer, and i max is defined as:
i max = z (α - f0) (3)
where α = end m/z. The resulting windows are set to a width based on the desired distance from the peptide ion center to accommodate for the resolution of the instrument. A 0.025 m/z distance has been shown to be effective for a resolution of ~60,000, that generates a baseline peak width of ~0.015 m/z. The Deuterator uses this number as default. Ions with m/z values outside of these sub-ranges cannot arise from the peptide ion of interest and thus they are removed from further consideration. The weighted average of the data within these expected m/z sub-ranges is then calculated. The interface also allows the limits of the m/z sub-ranges to be adjusted, which enables the correct peaks to be extracted from the spectral data. This approach has been shown to correctly distinguish peptides in complex situations such as overlapping peptides or peptide ions with poor signal-to-noise (S/N) ratios. The limits of m/z sub-ranges are displayed in the user interface as yellow bars, allowing the visual confirmation of the approach (Figure 4 bottom).
Main analysis page
It is often beneficial to visually validate and manually curate the data. The main analysis page provides the necessary tools to facilitate the validation, curation and recalculation of the %D and Centroid values. This page has three sections: the data grid, the spectral viewer, and the extracted ion chromatogram.
When a peptide row is selected from the data grid, the co-added spectral data for the calculated m/z range of the peptide are presented in the main spectral viewer (Figure 4). The best-fit theoretical isotopic envelope is then overlaid and the sub-ranges are presented for high-resolution data. The indicator bars display the mz0, mz100, m/z, and centroid range positions. Adjustment of the m/z range controls allow for zooming in and out of the spectral data. Navigation controls are available to further refine the sub-range widths (for high resolution data), retention time range, and centroid range of the selected peptide, with the %D and centroid values being automatically recalculated after each refinement. The extracted ion chromatogram (Figure 5) is also populated when a peptide is selected, and the averaged ion intensities for a given mass across all scans is presented. Blue bars display the retention time ranges used for the extracted ion plots, and can be adjusted for refinement of the scan range with the best abundance for a given peptide's centroid mass. For example, when the retention time start and end values are equal, the view will represent spectral data from the nearest single scan. When the user has completed data curation, the results can be saved.
Adjusting data in the main page can be a lengthy task, especially if there are a large number of curations needed for several experimental repetitions. Retention time and m/z ranges for like peptides across replicates are usually consistent. By synchronizing across the replicate data sets the number of manual curations needed is significantly reduced and automated reprocessing of centroid calculations is enabled. The synchronization page provides this functionality by allowing the selection of a "core' time point, a list of "sync" time points, and data column values. The process then adjusts the appropriate ranges and the centroids are recalculated for each peptide row. This has demonstrated to save substantial amounts of time by automating the curation process. A typical experiment produced approximately 100 peptides at eight timepoints. Each experiment was replicated four times for consistency. In this case 3200 peptides, each taking one minute to curate with the software, would cost the end user around 53 hours. In our tests, the synchronization function has been shown to be effective on roughly 20% of peptides saving over 10 hours of manual curation time.
The Deuterator in practice
The Deuterator has been used extensively on a number of projects over the last year, one of which has been published . Another example of these projects was the measurement of perturbation of H/D exchange rates for the nuclear receptor PPARγ upon binding of synthetic and endogenous ligands. The aim of this research was to establish the different mechanisms of activation of the receptor upon binding of full and partial agonists. The system has been tested with high and low-resolution raw mass spectral data from Thermo Electron LTQ Orbitrap (resolution ~60,000) as well as LTQ linear Ion-Trap (resolution ~1,500). The centroid calculation is a simple weighted average function, and so the validation of the centroid results has been straightforward. The software has been compared to existing tools such as Magtran  and the results have been shown to be identical. Native search engine data from the database search programs Mascot and Sequest, and X!Tandem have also been used with the system. The in-house installation is tightly integrated with commercial LIMS software (Proteus, Genologics, Canada), which captures the project files and streamlines automated processing.
Rendering components will be built that provide extended graphical capabilities, 2D and 3D color gradient maps, and data grids that convey changes in deuterium incorporation between different experiments. Sequence coverage can be presented in the user interface with heat maps, color-coding the rate of exchange. Improvements in the preprocessing phase will be implemented which will improve precision and reduce further the need for manual intervention. Ongoing tests to increase compatibility with other instrument data formats will be conducted. Any future developments to The Deuterator will be made accessible to the community.
The Deuterator is a web application presenting integrated and novel functionality in H/D exchange analysis and it is compatible with multiple file formats. The interface presents the centroid results and associated data for each peptide and allows for "drilling-down" and visualization of the mass spectral data. Developed to aid scientists at The Scripps Research Institute in the analysis of H/D exchange data, it is freely available online and eventually will be offered as a standalone open source solution.
Availability and requirements
Project name: The Deuterator
Project home page: http://deuterator.florida.scripps.edu
Operating system: Platform Independent
Programming language: Java/JSP
Other requirements: N/A
License: available free of charge to academic, government and non-profit users for non-commercial use
- H/D exchange:
Java Server Pages
- m/z :
Nuclear magnetic resonance
We would like to thank Robert Driscoll and Mohammad Fallahi for assistance with formulas and overall review of the manuscript. The Scripps Research Institute, Scripps Florida is supported by the State of Florida.
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