Hexicon 2: Automated Processing of Hydrogen-Deuterium Exchange Mass Spectrometry Data with Improved Deuteration Distribution Estimation

Abstract

Hydrogen–deuterium exchange (HDX) experiments analyzed by mass spectrometry (MS) provide information about the dynamics and the solvent accessibility of protein backbone amide hydrogen atoms. Continuous improvement of MS instrumentation has contributed to the increasing popularity of this method; however, comprehensive automated data analysis is only beginning to mature. We present Hexicon 2, an automated pipeline for data analysis and visualization based on the previously published program Hexicon (Lou et al. 2010). Hexicon 2 employs the sensitive NITPICK peak detection algorithm of its predecessor in a divide-and-conquer strategy and adds new features, such as chromatogram alignment and improved peptide sequence assignment. The unique feature of deuteration distribution estimation was retained in Hexicon 2 and improved using an iterative deconvolution algorithm that is robust even to noisy data. In addition, Hexicon 2 provides a data browser that facilitates quality control and provides convenient access to common data visualization tasks. Analysis of a benchmark dataset demonstrates superior performance of Hexicon 2 compared with its predecessor in terms of deuteration centroid recovery and deuteration distribution estimation. Hexicon 2 greatly reduces data analysis time compared with manual analysis, whereas the increased number of peptides provides redundant coverage of the entire protein sequence. Hexicon 2 is a standalone application available free of charge under http://hx2.mpimf-heidelberg.mpg.de.FigureᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s13361-014-0850-y) contains supplementary material, which is available to authorized users.

Analytical Aspects of Hydrogen Exchange Mass Spectrometry

“…Other reports over the years described using overlapping fragments to improve spatial resolution [e.g. (63, 77, 100, 101)] and a recent renaissance (102–104) has revived the idea but with the use of much improved computational and analytical tools. Using overlapping fragments is not without problems (105) so caution must be exercised.…”

“…In the absence of better separation or reduction to the complexity of the digestion, overlapping peptide signals have be deconvoluted by the mass spectrometer itself when there is high mass spectrometric resolving power [e.g., (119–123)] or with software methods [e.g., (104, 120, 124–127)]. These approaches, while valuable, do not solve the underlying issue of poor analytical performance under quench conditions.…”

HDX-MS guided drug discovery: small molecules and biopharmaceuticals

“…Differential HDX-MS analysis of a protein under different conditions (e.g apo vs. holo protein) has emerged as an important tool to probe the effects of chemical modifications, mutations, and binding events on protein stability and conformational dynamics (Figure 1). The development of fully automated HDX platforms with improved software has enabled the rapid collection and near real-time processing of data with statistical analysis, a critical advancement for the integration of HDX-MS into drug discovery programs [4-9]. Correlating deuterium incorporation patterns from several small molecule ligands with functional assays has proven to be an effective approach to develop structure activity relationship and delineate functional selectivity between closely related compounds [10-13].…”

HDX Match Software for the Data Analysis of Top-Down ECD-FTMS Hydrogen/Deuterium Exchange Experiments

“…Numerous approaches have been developed mainly for the processing of bottom-up HDX data, ranging from simple peak processing utilities [4] to fully integrated instrument-software platform solutions [3,[5][6][7]. Arguably, the most advanced approaches for determining deuteration levels combine the identification of the deuterated peptide (thereby determining its elemental composition), calculation of its deuterated isotopic distribution, and fitting it to the experimental data.…”