Capillary Electrophoresis Analysis of Proteins using Phospholipid Based Materials

Capillary electrophoresis has many advantages that make it a powerful technique for the analysis of proteins, which is challenging due to the natural heterogeneity of proteins. The small sample volume required for each analysis (nano- and pico-liter), fast separation times (\u3c30 \u3emin), and ability to couple the separation to mass spectrometry are all factors that make capillary electrophoresis an ideal technology for protein separations. Capillary zone electrophoresis and capillary gel electrophoresis are the two most commonly employed modes for protein analysis and allow for rapid, high resolution separations that can be automated. This makes capillary electrophoresis ideal for meeting the needs of protein analyses in the biopharmaceutical and clinical fields. Still, methods are continually improving in order to address the challenges of analyzing these samples with capillary electrophoresis, such as adsorption to the capillary wall, heterogeneity due to multiple isoforms, and maintaining native structure through analysis. The work presented utilized a coating and/or sieving matrix composed of the phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) to confront these needs. The phospholipids lead to reduced adsorption and enhanced resolution that can be tuned with temperature. Furthermore, the phospholipids have been shown to support and enhance protein function and lifetime in-capillary, which makes the material promising for native analyses of proteins in the future. The phospholipids were the basis of a hybrid coating using cetyltrimethylammonium bromide (CTAB) that enabled simultaneous separation of acidic and basic proteins. The semi-permanent coating cost less than $0.10 to apply to a traditional silica capillary, and unlike previously published work, the ability to analyze both basic and acidic proteins using the phospholipid-cetyltrimethylammonium bromide (CTAB coating) without requiring the use of low pH buffers that lead to denaturing proteins. The phospholipid coating reduced protein adsorption compared to bare fused silica; however, it also suppressed and reversed the electroosmotic flow. This led to acidic proteins being analyzed in one polarity while basic proteins were analyzed in another polarity. Incorporating the cetyltrimethylammonium bromide (CTAB) into the phospholipid coat generated sufficient electroosmotic flow for simultaneous analysis of both acidic and basic proteins. The coating was stable through extensive flushing and exhibited no protein adsorption through six consecutive analyses with no flushing in between. The coating was applied to the analysis of abundant proteins in human serum and the accurate quantification further demonstrates the lack of interaction between the surface and the proteins. For the first time, the phospholipid nanogel was utilized to add a size-based component to the separation mechanism of proteins. The phospholipid nanogel cost $0.14 per 5 μL, which is less than commercial matrices currently available, and when used in-capillary as described in this paper, the matrix utilized for one sample is only $0.04. Furthermore, the thermally-responsive viscosity of the nanogel makes it a more user-friendly material than traditional gels since it can be inserted into the capillary similarly to buffers at temperatures below 24°C without the requirement of high pressures or chemical measures for cross-linking in-capillary that can lead to non-uniform matrices. The temperature is then raised to create a gel-like matrix for the separation. As demonstrated in prior work with DNA, the concentration of the nanogel can be utilized to tune the resolution to the size range of the analytes of interest, so the concentration used is determined by the mass range relevant to the analyte. Because the application of this material for the separation of proteins is novel, the separation mechanism is investigated with focus being placed on the impact of phospholipid concentration. A size-based component is superimposed on the preexisting charge-to-size ratio mechanism using the phospholipid. This leads to enhanced the resolution of proteins in human serum and allows for the accurate quantification of the proteins using external standards