HDXFinder: Automated Analysis and Data Reporting of Deuterium/Hydrogen Exchange Mass Spectrometry
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Hydrogen/deuterium exchange in combination with mass spectrometry (H/D MS) is a sensitive technique for detection of changes in protein conformation and dynamics. However, wide application of H/D MS has been hindered, in part, by the lack of computational tools necessary for efficient analysis of the large data sets associated with this technique. We report a novel web-based application for automatic analysis of H/D MS experimental data. This application relies on the high resolution of mass spectrometers to extract all isotopic envelopes before correlating these envelopes with individual peptides. Although a fully automatic analysis is possible, a variety of graphical tools are included to aid in the verification of correlations and rankings of the isotopic peptide envelopes. As a demonstration, the rate constants for H/D exchange of peptides from rabbit muscle pyruvate kinase are mapped onto the structure of this protein.
Key wordsHydrogen/deuterium exchange Mass spectrometry Protein dynamics Fourier transform ion cyclotron mass spectrometry
There is a growing appreciation that hydrogen/deuterium exchange mass spectrometry (H/D MS) can provide insights into different aspects of protein conformation and dynamics. Thus, there is a current need for computational tools that can aid in the analysis of the very large data sets produced by these techniques. Efforts in program development to-date focus on isolated aspects of data analysis; examples include centroid calculation [1, 2], calculation of deuterium content in proteins or peptic peptides , and determining envelope data . These tools are useful for the statistical analysis, visualization, and plotting of deuterium content.
There are several problems associated with complex multidimensional H/D MS experiments that have not been fully addressed by the programs listed above. One is the detection of peptide ion isotopic envelopes, complicated by the shift in mass and complexity as a consequence of varying deuterium content. Another is the isolation of individual isotope envelopes from overlapping peptide ions. Programs such as Hydra , HeXicon , the Deuterator , and its successor, HD Desktop  attempt to address these problems using statistical methods for fitting experimental data to theoretical isotope envelopes. Unfortunately, these programs require, as initial input, the HPLC retention times of the peptide ions of interest. Thus, even with these aids, user input is needed at all phases of data interpretation, making this process time-consuming and prone to human error. There are also recent developments in the analysis of H/D MS data acquired on a 14.5 Tesla FT-ICR instrument , but these instruments are not commonly available.
Here we report HDXFinder, an application for peptide detection and for automatic peak assignments, which does not depend on HPLC retention times. Detection and isolation of isotope peptide envelopes, including extraction of overlapping peptide ions, are fully automatic functions performed by HDXFinder. The set of tools included in HDXFinder has been tested at a resolution power as low as 12,500 for an ion with m/z = 400.
2 Program Description and Results
2.1 Programming Background
HDXFinder is freely accessible from the KU Medical Center Mass Spectrometry Core Laboratory (https://HDXAnalyzer.kumc.edu/) to all not-for-profit users. Due to the recent use of the name “HDX-Analyzer” by another group, we have modified the name of the program described here from “HDXAnalyzer” to “HDXFinder.” Therefore, the address of the program (https://HDXAnalyzer.kumc.edu/) does not match the program name (HDXFinder).
2.2 Data Input and Analysis
Three sets of data are required as input for the program: (1) a data file(s) containing the mass spectrometric information, (2) the sequence of the protein of interest, and (3) a list of peptides generated from the protein of interest. Additionally, a pdb datafile can be uploaded for representation of the output data on the three-dimensional structure of the protein. HDXFinder can be used for data analysis of files generated by a variety of instruments, independent of manufacturer. Thus, it accepts data in the mzXML  or mzML  format, both developed to facilitate exchange of mass spectrometric information across multiple platforms. There are freely available converters for the specific data files generated by most manufacturers of mass spectrometers (http://tools.proteomecenter.org) .
The list of peptides of interest can be generated from any of the search engines broadly used for protein identification (a topic beyond the scope of this article). For each peptide included in this list, the following information must be included in a comma-separated (csv) format: the peptide sequence, the peptide mass (M + H+), and the peptide charge. For the experiments described here, the list of peptides of interest from the peptic digest of rabbit muscle pyruvate kinase was generated from Sequest (see Supplementary information).
2.3 Determining the Rate Constants of Exchange
Deuterium content is measured as the difference of average mass between the deuterated and the theoretical mass of the undeuterated peptide. Once data have been correlated and ranked, rate constants of exchange can be calculated for each peptide. Rate constants of exchange are determined by following the mass of the peptide as a function of time of the exchange reaction, using a single exponential fit. Users may also choose to export data to enable determination of the rate constants of exchange with other fit models.
3 Results and Output
HDXFinder works by first extracting all isotopic envelopes from an MS experiment. This process is automatic and is independent of any consideration for the nature of isotopic envelopes (i.e., chemical composition) or for their deuterium content. The only limiting variables considered are that the isotopic peptide envelopes should have a measurable charge (z = 1 to 6) and a chromatographic elution profile consistent with an eluting ion (see Supplementary material). There are several advantages to extracting all isotopic envelopes before correlating them to individual peptides: (1) the extraction of isotopic envelopes is performed only once, resulting in a significant increase in the overall speed of the analytical process. (2) The extraction of all isotopic envelopes present in a given experiment, independent of the identification of the corresponding amino acid sequence, allows for the reporting of the isotopic envelopes of unknown primary sequence. This may allow the user to increase protein coverage by re-attempting peptide identification using an inclusion list for these particular ions. (3) The application of the algorithms used for this process results in the extraction of overlapping isotopic envelopes and the removal of spurious noise.
To help prevent back exchange and maintain deuterium label, peptides are eluted quickly (i.e., steep gradients and high flow rates) from HPLC reversed-phase chromatographic columns. However, despite considerable effort in instrument and method development [13, 14, 15, 16], steep gradients often result in the co-elution of peptide ions with overlapping isotopic envelopes . This makes it difficult to determine accurately the experimental average mass of the peptides. As mentioned above, programs such HD Desktop  and HeXicon  determine average masses by performing curve-fitting of the isotopic envelopes to a theoretical envelope.
In contrast to other programs that incorporate an evaluation of HPLC retention times as a step for extracting average masses of peptides, HDXFinder does not require, as an input, the retention times for the detection of the eluting peptides. Instead, all isotopic peptide envelopes are detected automatically and subsequently matched and correlated to the peptides included in the list. The retention time along with the intensity of the ion, as measured by HDXFinder, are utilized for the ranking of ions (see Supplementary material for details).
In summary, HDXFinder provides a simple, fast, and automated routine for the identification of peptides in H/D MS. With these advantages, this program should find broad application in the mass spectrometry community.
The authors acknowledge support in part for this work by NIH grant DK78076 awarded to A.W.F. and by ITS grant from the University of Kansas to A.A.S. The authors thank Dr. David Nualart for valuable discussions.
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