Quantitating the Statistical Distribution of Deuterium Incorporation To Extend the Utility of H/D Exchange MS Data

Abstract

Measuring the statistical distribution of deuterium incorporated into enzymatically derived peptide fragments provides a valuable dimension to hydrogen/deuterium exchange mass spectrometry data. In this paper, we will discuss our improvement to the linear least-squares method for determining this distribution, through the addition of "zeroes" to the end of the deuterated isotopic envelope, to partially compensate for data truncation due to finite instrumental signal-to-noise ratios. The value of the distribution is demonstrated in a simple experimental example, where the linearity between average deuteration and percent D2O used to label test peptides hides a more complex relationship between the site-labeling probability and the total number of sites. This method offers the opportunity to resolve cases where a single peptide experiences distinct, independent biochemical states with each bearing a unique average deuteration; this can occur when a protein is modified to substoichiometric levels. From the experimentally determined distribution of a heterogeneously deuterated peptide, it was possible to extract the average deuteration of each component of the mixture.

Automated Data Extraction from In Situ Protein-Stable Isotope Probing Studies

“…Statistical analysis of label distribution, originally described by Chik et al, 31 was implemented in C# and added to the DeconTools Framework as part of this work. The software is open-source and available at http://omics.pnl.gov/software.…”

Improved Sequence Resolution by Global Analysis of Overlapped Peptides in Hydrogen/Deuterium Exchange Mass Spectrometry

“…We have an option of estimating the rates for these "fast" and "slow" residues in the initial search of first and final incubation points, after which their rates are allowed to vary ± 25 %, but in practice the program is fast enough to float even those residues. The black bars on top of Figure 2b denote five groups of linked residues (3, 4), (6,7), (9)(10)(11)(12)(13)(14)(15), (16,17), and (27-29) for which we can determine the individual rate constants but cannot assign them to specific residues.…”

“…The simplest algorithm provides a fast and automated way to calculate the average mass of each proteolytic peptide [3]. The most common tools employ deconvolution of the isotopic distributions to extract deuterium incorporation based on natural isotopic abundance [4][5][6][7]. A different approach based on a peak width method allows the characterization of HDX kinetics [8].…”

Semi-automated data processing of hydrogen exchange mass spectra using HX-Express

“…on the same plot to aid in comparison. While other HXMS data analysis software exists [3][4][5] HX-Express is available as freeware and runs within the ubiquitous Microsoft Excel environment.…”