Structural Proteomics Takes Aim at Disease

Unlike the genome, which is relatively static, the proteome is dynamic, constantly changing in response to intracellular and extracellular environmental signals. Modern mass spectrometric techniques, electro-spray ionization and matrix assisted laser desorption ionization, are capable enough to take the “molecular elephants” of the proteome into gas phase, thereby studying them individually and collectively in hetero-molecular assemblies (1, 2). 

This has huge implications for medicine. In patients, a variety of factors have a bearing on protein expression and post-translation modification, including the disease itself, pharmacological interventions, genetic factors and environmental variables. High throughput analysis of the proteome in a limited clinical sample is already feasible and ongoing improvements to increase resolution, sensitivity, mass accuracy and analysis speed of mass spectrometers along with the advent of higher order dimensions in separation will permit proteomic analysis of virtually any challenging clinical sample in a single step in the near future.

So what is the next quantum step?  Proteomics need not be restricted to a study of primary structure. It can be extended to assess the thermodynamic stability of the three-dimensional structure of proteins, finally to arrive at an understanding of molecular mechanisms through structure-function correlation. 

The discovery of protein structures has – since alterations in the chemical structure of hemoglobin in sickle cell anemia was achieved in the 1950s (3) – made it possible to assign disease to the functional disorder of a few atoms. However, structural analysis has been confronted with three issues. First, using any spectroscopic tool has required pure protein in large quantities, which is difficult to achieve with clinical samples. Second, due to complex milieu of molecules in the cell, in vitro stability parameters might not be exactly the same as those that occur in vivo.

And third, most spectroscopic methods that monitor molecular properties are not molecule-specific; rather, they are functional group-specific making it impossible to have structure-function analysis at the residue levels of a protein in vivo.

We believe that the solution to these problems lies with hydrogen deuterium exchange-based mass spectrometry (HDX-MS). By exploiting the permeability of heavy water (D2O) through cell membranes, HDX-MS has been used to measure the thermodynamic stability of intact protein structure in vivo (4). HDX-MS has also been applied in vitro to establish structure-function correlations at the residue level of proteins in non-covalently bonded complexes by studying the kinetics of isotope-exchange patterns (5).

Ion mobility spectrometry (IMS) adds a further dimension to structural proteomics research by separating molecules on the basis of shape. The combination of HDX-MS and IMS can be used to explore the underlying mechanisms of protein folding and  protein-ligand interactions in normal and disease situations. In our view, an investigation of protein function through structure-function correlation in vivo using isotope exchange-based mass spectrometry would be an important scientific breakthrough, one that connects us back to the origins of molecular medicine with the discovery of the molecular basis of sickle cell anemia in 1949 (6).