Capillary electrophoresis separations play an important role in challenging bioanalyses.  With new materials and strategies, this separation technique is prominent in next generation therapeutics and in shedding light on physiological systems.  This presentation will reveal the role of biocompatible nanomaterials and instrumental advances to leverage the high efficiency and throughput of capillary separations.  Advances in this technology overcome barriers to universally analyzing proteins, which are among the most diverse class of physiologically relevant biomolecules.  In particular, protein isoforms and post-translational modifications will be addressed.  A rapid, inexpensive, and automated method is demonstrated to successfully quantify modifications with N-glycans (e.g. fucosylation, sialylation, branching, and bisection), which are a result of both normal and aberrant physiological processes.  With this advance subtle differences in proteins can be distinguished.  The separation is facilitated with self-assembled nanogels in a capillary that also contains stationary zones of lectins and/or enzymes.  The nanogel electrophoresis generates separation efficiencies of 500,000 plates and easily resolves positional isomers.  Because this technology is biocompatible, native analyses are also feasible.  The nanomaterial additives used for these separations have a thermally dependent viscosity, which provides a means to precisely control and position specific zones of enzymes and lectins within the capillary to both process and separate proteins in nanoliter volumes in an automated instrument.  The nanogel supports successive reaction in a channel (i.e. in series) because the viscosity of nanophases minimizes dispersion.  Each molecular affinity step of the analysis utilizes a small (~5 nL) re in the channel.  Nanogel preparations are inexpensive, costing $0.09 for 5 mL.  The structural features of proteins are identified and quantified without the need for standards.  Moreover, the nanophases are compatible with UV absorbance, fluorescence, and mass spectrometry detection.  Cost-effective and rapid monitoring of glycosylation is critical to ensuring the efficacy of protein-based antibody drugs and to elucidating the role of N-glycans in physiological processes.

Capillary electrophoresis separations:  New tools to advance biotechnology

ACS Fall 2021

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