Ernst Althaus

Hydrogen-deuterium exchange (also called H-D or H/D exchange) is a chemical reaction in which a covalently bonded hydrogen atom is replaced by a deuterium atom, or vice versa. Usually the examined protons are the amides in the backbone of a protein. The method gives information about the solvent accessibility of various parts of the molecule, and thus the tertiary structure of the protein.
In modern times H/D exchange has primarily been monitored by the methods: NMR spectroscopy and mass spectrometry. Each of these methods have their advantages and drawbacks. The main disadvantage of mass spectrometry is that one obtains exchange data for peptic fragments and assigning exchange speeds to single residues has to be done by manual interpretation.
We provide an automated method to resolve this problem. More precisely, we present an algorithm that enumerates all possible exchange speeds for single residues that explain the observed data of the peptic fragments. As the number of possibilities is often very large, we combine sets of as- signments to equivalence classes which are easily interpreted such that the number of equivalence classes is typically very small.
The assignment of exchange speeds to single residues from the data of the peptic fragments is a combinatorial problem. Hence, we applied methods from combinatorial optimization to it, i.e. we show how to formalize the problem into an integer linear program and propose a method to solve the problem.

The experiment

The experiment is initiated by dilution of the protein solu tion into a biological buffer made with D2 O. Solvent accessible hydrogens are exchanged with deuterium. The exchange is quenched (greatly slowed) by dropping the pH to pH 2.3 and lowering the temp to 0-4° degree C. The protein complex is digested with pepsin or protease type XIII and on-line liquid chromatography is performed directly to the FT-ICR MS (Figure 1). Deuterium incorporation is monitored by the increase in mass of each peptic fragment as the deuteron is added.

Figure 1: Schematic of the HDX experiment.
The data sets produced are large and each spectrum has hundreds of overlapping peptic fragments. From this data, the exchange rate is easily determined for the same peptic fragments from protein and protein complex. When peptic fragments are not directly comparable, but are overlapping (Figure 2) manual interpretation must be performed to assign exchange speed to single residues. HDX data analysis is the greatest bottle-neck in these experiments, thus automated data analysis is necessary.

Figure 2: Schematic of the HDX experiment.