Optimizing capillary electrophoresis for top-down proteomics of 30-80 kDa proteins.

The direct analysis of intact proteins via mass spectrometry offers compelling advantages in comparison to alternative methods due to the direct and unambiguous identification and characterization of protein sequences it provides. The inability to efficiently analyze proteins in the ‘middle mass range’, defined here as proteins from 30-80 kDa, in a robust fashion has limited the adoption of these “top-down” methods. Largely a result of poor liquid chromatographic performance, the limitations in this mass range may be addressed by alternative separations that replace chromatography. Herein, the short migration times of capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) have been extended to size-sorted whole proteins in complex mixtures from Pseudomonas aeruginosa PA01. An electrokinetically pumped nanospray interface, a coated capillary and a stacking method for on-column sample concentration were developed to achieve high loading capacity and separation resolution. We achieved full width at half maximum of 8-16 seconds for model proteins up to 29 kDa and identified 30 proteins in the mass range of 30-80 kDa from Pseudomonas aeruginosa PA01 whole cell lysate. These results suggest that CZE-ESI-MS/MS is capable of identifying proteins in the middle mass range in top-down proteomics.This work was supported by the National Institute of General Medical Sciences and National Institute of Drug Abuse of the National Institutes of Health under award numbers R01 GM067193 and DA018310, and the National Science Foundation under award number ABI-1062432 to Indiana University. Additional support was provided by the Chicago Biomedical Consortium with support from The Searle Funds at The Chicago Community Trust, and the Robert H. Lurie Comprehensive Cancer Center