G-quartet DNA stationary phases for capillary electrochromatographic separation of proteins

The development of automated processes that provide sensitive, rapid, and non-denaturing separations of a wide variety of proteins is becoming increasingly important in agriculture, industry, and medicine. Towards this end, G-quartet-forming oligonucleotides were investigated as stationary phases for protein separations by open-tubular capillary electrochromatography (OTCEC). A variety of protein systems, including bovine milk proteins, human cell lysates, and human serum, were utilized to study the potential of these novel stationary phases.; Separations of two genetic variants of bovine β-lactoglobulin, which differ by only two amino acid residues, were achieved using G-quartet stationary phases but not using either a bare capillary or a capillary coated with a non-G-quartet-forming oligonucleotide. Separation was also achieved using a capillary coated only with the linker molecule, but very strong protein-linker interactions were observed. These results suggest that the G-quartet stationary phases may result in less denaturing separation than is commonly achieved using hydrocarbon-based stationary phases.; The eight major proteins in bovine milk were also separated using this approach, without the traditional, harsh sample pre-treatments. Better resolution was achieved using the G-quartet-coated capillaries than was achieved using either a bare capillary or a non-G-quartet-forming oligonucleotide-coated capillary. A four-plane G-quartet stationary phase was able to resolve three peaks for α-casein, attributed to α_s2-casein and two genetic variants of α_s1-casein. It was also possible to detect thermal denaturation of the proteins in the milk sample using the four-plane G-quartet stationary phase. These results suggest that the G-quartet stationary phases could be used to separate very similar protein structures, such as those arising from genetic variations or post-translational modifications.; Separations of complex biological samples were performed to investigate the potential of the G-quartet DNA stationary phases for use in multi-dimensional proteomic analysis. Results demonstrate potential of this technique for identification of a unique biomarker in lung cancer, and for incorporation into a two-dimensional separation. With the ability to be directly interfaced with sensitive modes of detection and the demonstrated ability of the G-quartet DNA stationary phases to achieve rapid, reproducible, and possibly non-denaturing protein separations, this approach could prove valuable in both agriculture and the complex field of proteomics.