OligoR: A Native HDX/MS Data Processing Application Dedicated to Oligonucleotides

Hydrogen–deuterium exchange mass spectrometry (HDX/MS) is increasingly used to study the dynamics of protein conformation. Coupled to native MS, HDX can also characterize the conformations of oligonucleotides and their binding to cations, small molecules, and proteins. Data processing and visualization of native HDX/MS of oligonucleotides requires dedicated software solutions. OligoR is a web-browser-based application that addresses the specific needs of DNA HDX/MS and native MS experiments from raw data in an open format to visualization and export of results. Whole experiments spanning many time points can be processed in minutes for several mass-separated species. To access valuable folding dynamics information, we have developed a simple and robust approach to deconvolute bimodal isotope distributions, even when they are highly overlapping. This approach is based on modeling physically possible isotope distributions determined from chemical formulae and could be extended to any type of analyte (proteins, peptides, sugars, and small molecules). All results are presented in interactive data tables, and publication-quality figures can be generated, customized, and exported.


Binding kinetics
The binding kinetics of K + (500 µM) with 23TAG (10 µM) was recorded on a Thermo Orbitrap Exactive mass spectrometer for 30 minutes. Raw data was converted to the mzML format as described above. Data was analyzed in OligoR by selecting the species of interest (complex with 0, 1 and 2 K + for the 5charge state).

Gaussian fitting
Fitting of isotopic distribution with Gaussians was performed with Equation S1, where , and are respectively the area of the peak (reflecting the abundance of the isotopic distribution), position of the center of peak (centroid of the isotopic distribution) and full width at width at half maximum of the peak ( = 2 × √2ln(2) × , where is the standard deviation).
The estimate of overlap  between isotopic distributions was calculated with the overlapTrue function from the overlap R package. 2

Titration experiments
The determination of response factors and from titration experiments was implemented in R following our previously published method. 3 The calculation of Moore-Penrose generalized inverse of matrices was implemented using the gnm package.

. Generation of bimodal spectra
Bimodal spectra were generated by convoluting two isotopic distributions generated by OligoR, specifically with the function peak.positionR, which takes into account the oligonucleotide sequence, the number of exchangeable sites, and the solution deuterium content. For each bimodal spectrum, the abundances were randomized and the deuterium content DCi of a population i was set as an integer between 9 and 50 (in %), with DC2 > DC1 (to discard duplicates) and a maximum difference (DC2 -DC1) of 30 to only obtain spectra with some degree of overlap. The application of this approach to three sequences T30177-TT, 23TAG and VEGF binding 2 K + at z = 4-generated 2181 bimodal spectra with overlaps ranging from 0.03 to 0.94. Figure S6. Heatmap of the difference in deuterium content between the two generated populations (left) and corresponding overlaps for [23TAG•2K + ] 4-(right). Each tile corresponds to a bimodal spectrum, and this approach was applied to three sequences.
Noise was added on the y axis using a random generation function (rnorm, from the stats package for R) for the normal distribution with the standard deviation set to 0.01.

Results
The fitting algorithm of OligoR was then applied on all spectra. A few randomly sampled examples are shown below.

S12
The mean squared error (MSE), centroid error, and isotopic population abundance error were plotted as a function of the overlap between the isotopic population.