Considerable interest exists in endogenous peptides as potential biomarkers, since they act as signaling molecules and are formed by degradation of proteins. A crucial step in the profiling of these peptides is the sample preparation, which aims to enrich the low-abundant peptides, while removing interfering matrix compounds. In a feasibility study we examined the suitability of electrodialysis (ED) for this purpose. A custom-made device was developed from the low-binding material Kel-F. It consisted of two compartments separated by a dialysis membrane, over which a voltage was applied. One compartment served as donor (containing the sample), while the smaller acceptor compartment collected the peptides. The pr... More
Considerable interest exists in endogenous peptides as potential biomarkers, since they act as signaling molecules and are formed by degradation of proteins. A crucial step in the profiling of these peptides is the sample preparation, which aims to enrich the low-abundant peptides, while removing interfering matrix compounds. In a feasibility study we examined the suitability of electrodialysis (ED) for this purpose. A custom-made device was developed from the low-binding material Kel-F. It consisted of two compartments separated by a dialysis membrane, over which a voltage was applied. One compartment served as donor (containing the sample), while the smaller acceptor compartment collected the peptides. The procedure was optimized by investigating the effect of the applied voltage, ammonium acetate buffer concentration, and ED duration using model peptides. Optimum conditions were found at 300 V (150 V/cm), 25 mM ammonium acetate buffer (pH 3.8) containing 20% v/v DMSO, and 10 min, respectively. With these optimized parameters, recoveries for the model peptides were found to be 35–85% (average 64%). Additionally, ED was successfully applied to the challenging synovial fluid biological sample (due to its high viscosity). In a synovial fluid sample from a rheumatoid arthritis patient, 27 peptides originating from 12 proteins were identified, of which a considerable fraction was not identified before with other methods. This demonstrates the usefulness and complementary nature of combining ED with nanoLC-MS for biomarker discovery. These results indicate that ED is promising as a fast and selective sample preparation method for the profiling of endogenous peptides.
