Surface-enhanced laser desorption-ionization retentate chromatography mass spectrometry (SELDI-RC-MS): a new method...

Protein biochip arrays carrying functional groups typical of those employed for chromatographic sorbents have been developed. When components of a protein mixture are deposited upon an array's functionalized surface, an interaction occurs between the array's surface and solubilized proteins, resulting in adsorption of certain species. The application of gradient wash conditions to the surface of these arrays produces a step-wise elution of retained compounds akin to that accomplished while utilizing columns for liquid chromatography (LC) separations. In retentate chromatography-mass spectrometry (RC-MS), the "retentate" components that remain following a wash are desorbed and ionized when a nitrogen laser is fired at discrete spots on the array after treatment with a laser energy-absorbing matrix solution. Ionized components are analyzed using a time-of-flight mass spectrometer (TOF MS). The present study demonstrates that protein biochips can be used to identify conditions of pH and ionic strength that support selective retention-elution of target proteins and impurity components from ion-exchange surfaces. Such conditions give corresponding behavior when using process-compatible chromatographic sorbents under elution chromatography conditions. The RC-MS principle was applied to the separation of an Fab antibody fragment expressed in Escherichia coli as well as to the separation of recombinant endostatin as expressed in supernatant of Pichia pastoris cultures. Determined optimal array binding and elution conditions in terms of ionic strength and pH were directly applied to regular chromatographic columns in step-wise elution mode. Analysis of collected LC fractions showed favorable correlation to results predicted by the RC-MS method.