Site-specific HDX-MS for increased resolution

Most methods to characterize protein-ligand binding interactions have drawbacks. Crystallography, obviously, requires your protein to crystallize, while NMR and cryo-EM both have size limitations and are resource-intensive. Hydrogen-deuterium exchange mass spectrometry (HDX-MS), which we wrote about earlier this year, is theoretically faster and can apply to more proteins, but the technique provides limited resolution. Now a new open-access paper in J. Am. Chem. Soc. by Derek Wilson and collaborators at York University, the Structural Genomics Consortium, and SCIEX have improved the resolution down to single amino acids.

 

As we wrote in March, HDX-MS relies on the exchange between deuterium and protons on proteins; slower exchange is associated with more stable secondary structures. The exchange is detected by digesting the protein with proteases and determining the mass of the resulting peptide fragments using MS; incorporation of deuterons increases the mass of the peptides. If, for example, a peptide has three deuterons incorporated, the mass would increase by 3. However, in conventional HDX-MS, one can’t tell exactly where the deuterons have exchanged.

 

Last year the researchers described a method in J. Am. Soc. Mass Spectrom. to do just this using some clever mass spectrometry experiments. I won’t go into details as the paper is open access, but suffice it to say that it involves electron-activated dissociation (EAD) to fragment the peptides while preventing the scrambling of the protons and deuterons that normally limits resolution.

 

In the new paper, the approach was applied to the oncology target WDR5, which we previously discussed here. The researchers applied site-specific HDX-MS to three small molecule and two peptide ligands. All five of these ligands had previously been characterized by conventional HDX-MS as well as surface plasmon resonance (SPR), and four had also been studied crystallographically.

 

Reassuringly, the results from site-specific HDX-MS were mostly consistent with conventional HDX-MS, but there were some surprises. For example, the site-specific approach revealed protection from exchange for one amino acid (which makes a hydrogen bond to the ligand) and enhanced exchange for the adjacent amino acid on the same peptide. In conventional HDX-MS, the “peptide contains offsetting uptake increases and decreases, resulting in a ‘net-zero’ signal.”

 

The researchers also found that, for another residue, the change in HDX-MS signal correlates with the affinity of the ligand, as assessed by SPR. The mechanism is unclear since the concentrations of ligands were far higher than their affinities, but the results are intriguing.

 

The new paper describes just a single, well-behaved target, but if site-specific HDX-MS applies more broadly, it would offer a significant advance in obtaining structural information. The researchers note that the data analysis is currently “manual and prohibitively cumbersome,” a problem that should be solvable with automation. I look forward to seeing it applied prospectively to more targets, particularly if it works for weak binders such as fragments.