| Summary: | 碩士 === 國立中山大學 === 化學系研究所 === 96 === The simultaneous separation of anionic and cationic proteins has been achieved by addition of high concentration of poly(diallyldimethylammonium chloride) (PDDAC) in capillary electrophoresis. A capillary was filled with PDDAC so that it would act as ion-pair reagents in the separation of anionic proteins. On the other hand, the PDDAC can also be used as coating additives for the analysis of cationic proteins. Increasing the concentration of PDDAC in the separation buffer had the ability to improve the separation efficiency, change the electrophoretic mobility, and alter the separation selectivity; however, this was not true in the case of analyzing proteins by using the PDDAC larger than 1.6%. By both using a buffer containing 1.6% PDDAC and applying pH-stepwise techniques, 13 proteins with a wide range of pI (4.7–11.1) and molecular masses (6.5–198.0 kDa) could be separated within 30 min in a single run. In addition to this separation, we observed notonly more peaks from alph-chymotrypsinogen A and aprotinin but also the bovine serum albumin (BSA) dimer and trimer. The second part describes a method for enrichment and separation of acidic and basic proteins using the centrifugal ultrafiltration followed by polyelectrolyte-filled capillary electrophoresis. In order to improve stacking and separation efficiencies of proteins, the separation buffer containing 1.6% poly(diallyldimethylammonium chloride) was added with gold nanoparticles (AuNPs), poly(ethylene oxide), cetyltrimethyl ammonium bromide, and poly(vinyl alcohol). As a result, the use of AuNPs as additives exhibited better efficiency in separation, stacking and analysis time. Even for large-volume samples (110 nL), the separation efficiencies of acidic and basic proteins remained greater than 104 and 105 plates/m, respectively. To further enhance detection sensitivity, protein samples were enriched using the centrifugal ultrafiltration, followed by our proposed stacking method. As a result, the detection sensitivity was improved up to 314-fold as compared with normal hydrodynamic injection. Additionally, the limits of detection at a signal-to-noise of 3 for most proteins are down to nanomolar range. We have validated the application of our method by means of analyses of 50 nM lysozyme in saliva samples. The proposed method was also successfully applied to the analyses of egg-white proteins, which have large differences in molecular weight and pI.
|
|---|
