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. 2016 Nov 15;32(22):3413-3419.
doi: 10.1093/bioinformatics/btw420. Epub 2016 Jul 13.

MEMHDX: an interactive tool to expedite the statistical validation and visualization of large HDX-MS datasets

Véronique Hourdel  1 , Stevenn Volant  2 , Darragh P O'Brien  1 , Alexandre Chenal  1 , Julia Chamot-Rooke  1 , Marie-Agnès Dillies  2 , Sébastien Brier  1
Affiliations

MEMHDX: an interactive tool to expedite the statistical validation and visualization of large HDX-MS datasets

Véronique Hourdel et al. Bioinformatics. .

Abstract

Motivation: With the continued improvement of requisite mass spectrometers and UHPLC systems, Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) workflows are rapidly evolving towards the investigation of more challenging biological systems, including large protein complexes and membrane proteins. The analysis of such extensive systems results in very large HDX-MS datasets for which specific analysis tools are required to speed up data validation and interpretation.

Results: We introduce a web application and a new R-package named 'MEMHDX' to help users analyze, validate and visualize large HDX-MS datasets. MEMHDX is composed of two elements. A statistical tool aids in the validation of the results by applying a mixed-effects model for each peptide, in each experimental condition, and at each time point, taking into account the time dependency of the HDX reaction and number of independent replicates. Two adjusted P-values are generated per peptide, one for the 'Change in dynamics' and one for the 'Magnitude of ΔD', and are used to classify the data by means of a 'Logit' representation. A user-friendly interface developed with Shiny by RStudio facilitates the use of the package. This interactive tool allows the user to easily and rapidly validate, visualize and compare the relative deuterium incorporation on the amino acid sequence and 3D structure, providing both spatial and temporal information.

Availability and implementation: MEMHDX is freely available as a web tool at the project home page http://memhdx.c3bi.pasteur.fr CONTACT: marie-agnes.dillies@pasteur.fr or sebastien.brier@pasteur.frSupplementary information: Supplementary data is available at Bioinformatics online.

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Figures

Fig. 1.
Fig. 1.
The MEMHDX strategy. (A) .csv file containing deuterium uptake values for all identified peptides is exported after raw data extraction and analysis by dedicated HDX-MS software (e.g. DynamX). The .csv file is uploaded to MEMHDX where a linear mixed-effects model is applied to statistically validate the dataset. Main results are displayed on a ‘Logit’ representation (for data clustering and validation) and visualized using a global summary plot and the 3D structure, where available. A user-friendly ShinyInterface facilitates the use of the application (Color version of this figure is available at Bioinformatics online.)
Fig. 2.
Fig. 2.
Example of statistical results generated with MEMHDX. (A) ‘Logit’ plot obtained with RD. The effect of calcium binding on the deuterium uptake behavior was measured on 162 RD peptides. Each dot corresponds to one unique peptide. Peptides are classified and color-coded based on their respective HDX behavior: peptides showing dynamic events in both the Apo- and Holo-state are colored in gray; peptides only dynamic in the Apo- or Holo-state are colored in red and blue; non-dynamic peptides in both states are colored in green. The statistical significance threshold was set to 1%. (B) Deuterium uptake curves for selected peptides in the Apo- (open circles) and Holo- (filled squares) states. The position of each in the ‘Logit’ plot is also reported (Color version of this figure is available at Bioinformatics online.)
Fig. 3.
Fig. 3.
HDX-MS results visualized by MEMHDX. (A) Global visualization plots of RD in both the Apo- and Holo-state. The relative fractional uptake values are plotted as a function of peptide position. This representation allows the user to visualize the deuterium uptake behavior of each peptide across the entire protein sequence. (B) Fractional uptake difference plot showing the variations of deuterium uptake between Apo- and Holo-RD. A high-uptake difference value corresponds to a large calcium-induced protective effect, while a low value is indicative of a weak effect. Significant peptides are highlighted in gray. The statistical significance threshold was set to 1%. (C and D) Mapping of the HDX results on the model of RD using the 3D-structural tool of MEMHDX. RD is shown as a cartoon or in a space filling model. Regions experiencing deuterium uptake or dynamic changes upon calcium binding are colored in cyan; regions with no change are colored in red (Color version of this figure is available at Bioinformatics online.)
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