In-Capillary Protein Enrichment and Removal of Nonbuffering Salts Using Capillary Electrophoresis with Discontinuous Buffers

Salt is abundant in biological samples and can cause problems in capillary electrophoresis (CE) due to excessive Joule heating and electrodispersion. Desalting with solid phase minibeds is currently most compatible with the small sample volumes of CE. They are however difficult to prepare and suffer from poor bed-to-bed reproducibility. Alternatively, enrichment of proteins and peptides was developed using CE, by trapping them at their isoelectric points with a discontinuous buffer of mismatched pH. Ionic salts, such as sodium chloride, do not possess isoelectric points and therefore are not retained by the discontinuous buffer. In this work, the removal of ionic salt during protein enrichment using CE with discontinuous buffers was investigated. Nonbuffering ions were found to electromigrate through the pH junction without disrupting the enrichment process and were eventually removed from the capillary. Mass spectral data obtained from the enriched and desalted sample confirmed a significant signal enhancement. Finally, a strong acid was introduced to remove the pH junction and thus facilitated a subsequent capillary zone electrophoresis separation. An integrated procedure of enrichment, desalting, and separation was demonstrated on a mixture of three protein standards.