Improvements in Capillary Electrophoresis-Based Analysis of Biomacromolecules

Abstract

Biomacromolecules, including proteins, polysaccharides, nucleic acids, and lipids represent a range of large, structurally diverse molecules that play integral roles in virtually every cellular process. Analyzing and understanding these molecules is crucial, especially in regards to their roles in human health, disease, and potential therapies but their complexity, diversity, and size make this task difficult. The range of separation modes and detection methods available in capillary electrophoresis (CE) provides a level of tunability and flexibility that couples well with the demands of complex biological separations. The goal of this thesis is to present advances in CE separations of two of these classes of biomacromolecules, specifically a CE-based Western blotting method that utilizes inkjetting to deposit protein samples for offline immunoassay as well as labelling and analysis of intact pneumococcal polysaccharides. Western blotting is one of the most common laboratory methods used for protein detection and relative quantitation. It is a widely used, two-stage assay that combines a size-based separation with an antibody affinity assay but suffers from long analysis times, large sample and reagent use, and manually intense processes. Here, a capillary-based system for Western blotting is described and evaluated. Sodium dodecyl sulfate-protein complexes are separated via capillary gel electrophoresis and then captured on a nitrocellulose membrane via inkjet deposition. Samples containing a combination of protein ladder and lysate were separated and captured on membrane in 15min compared to conventional Western blots which typically separates and electroblots samples in 1h–overnight. Ladder and specific protein signals were simultaneously detected in separate channels and provided for direct size comparison. This system decreases total protein requirements to 50–100ng, compared to the 1–50μg routinely used in conventional Western blots. Evaluation of separation voltage, sample collection speed, and distance between capture membrane and inkjet dispenser was performed. Limits of detection (LoDs) for actin were determined to be 8pM or an estimated 5fg total protein injected. The sample volume requirements of the system were further decreased to allow for sampling from as little as 2.5μL by altering the sample inlet and was applied to analyze small groups of islets of Langerhans, which are pancreatic micro-organs that secrete hormones to maintain blood glucose levels and whose dysfunction leads to diabetes. Analysis of proteins in islets is of interest for diabetes research. The CE-Western blot method was used to detect several proteins related to islet growth and metabolism (4E-BP1, p-4E-BP1, 4E-BP2, actin, Akt, AMPKα, ERK1/2, GSK-3α, GSK-3β, p-GSK-3β, p-mTOR, rpS6, p-rpS6, Stat3, Stat5, p-TSC2, α-tubulin, and IRS1) from samples containing three islets each and achieved signal-to-noise ratios up to 75x greater that those obtained from conventional Western blots using equivalent numbers of islets. Finally, CE with laser-induced fluorescence was explored for the analysis of intact pneumococcal polysaccharides. Streptococcus pneumoniae are bacteria capable of causing several diseases and have almost 100 known serotypes, each with a unique capsular polysaccharide. Modern pneumococcal vaccines utilize polysaccharides conjugated to a carrier protein but their efficacy is decreased by the presence of unconjugated polysaccharides – demonstrating a need to separate and quantify the free polysaccharide in vaccine products. Here, pneumococcal polysaccharides were labelled with 8-aminopyrene-1,3,6-trisulfonic acid in 30min. Monovalent solutions of nine representative pneumococcal polysaccharides were analyzed with LoDs of 0.27–1.88nM and r2>0.9911 achieved for all tested polysaccharides.