Use of Displacement Chromatography for Proteomic Applications (with Professor Platt in the Chemistry Department)

The field of proteomics, with the ability to rapidly and unequivocally identify complex protein mixtures from a diverse array of sample types (bacterial, plant, human, etc.), represents one of the most important bioanalytical challenges of our time. In recent years, proteomics has had a tremendous positive influence on both fundamental biological research and clinical diagnostics. The field has relied upon two major enabling technologies: mass spectrometric detection and upstream separation technologies capable of fractionating thousands of peptide/protein species. Although mass spectrometry has seen several recent advancements, there is still an urgent need to develop more effective bioseparation techniques for proteomic applications. We have been actively involved in both theoretical and experimental studies on ion exchange protein displacement chromatography. A steric mass-action (SMA) model of nonlinear ion-exchange chromatography has been developed which has made significant strides in understanding some of the complexities of ion-exchange displacement separations. We are currently carrying out a detailed parametric investivation to determine the influence of various displacers, displacer concentrations, stationary phases, and mobile phase conditions to optimize these difficult separations.

Representative Abstract:
"Use of displacement chromatography for proteomic applications." Steven T. Evans, Alex Freed, Mark D. Platt, and Steven M. Cramer. 234^th National American Chemical Society Meeting and Exposition, Oral Presentation, Boston, MA (2007).

Proteomics provides one of the most important bioanalytical challenges of our time. Displacement chromatography has significant potential for the simultaneous concentration and purification of complex biological mixtures. Model studies were carried out using high affinity, low molecular mass displacers for the ion exchange displacement chromatography of proteins and peptides to determine the limits of this approach. Parametric studies with various displacers, displacer concentrations, stationary phases, and mobile phase conditions were performed to optimize these difficult separations. Results indicate high affinity displacers employed at relatively low concentrations can effect high resolution separations amenable to the identification of low abundance solutes. Displacement ion exchange systems were then used in concert with nano-flow reversed phased liquid chromatography and mass spectrometry to evaluate the utility of this combined approach for the analysis of complex biological mixtures with a wide dynamic range of feed concentrations.