Simultaneous Preconcentration and Separation of Native Protein Variants Using Thermal Gel Electrophoresis

Proteins must maintain proper folding conformations and express the correct post-translational modifications (PTMs) to exhibit appropriate biological activity. However, assessing protein folding and PTMs is difficult because routine polyacrylamide gel electrophoresis (PAGE) methods lack the separation resolution necessary to identify variants of a single protein. Additionally, standard PAGE denatures proteins prior to analysis precluding determinations of folding states or PTMs. To overcome these limitations, a microfluidic thermal gel electrophoresis platform was developed to provide high-sensitivity, high-resolution analyses of native protein variants. A thermally reversible gel was utilized as a separation matrix while in its solid state (30 degrees C). This thermal gel provided sufficient separation resolution to identify three variants of a fluorescently labeled model protein. To increase detection sensitivity, analyte preconcentration was conducted in parallel with the separation. Continuous analyte enrichment afforded detection limits of 500 fg of protein (250 pM) while simultaneous baseline separation resolution was achieved between variants. The effects of temperature on thermal gel electrophoresis were also characterized. The unique temperature-dependent outcomes illustrated how method performance can be tuned through a thermal dimension. Ultimately, the high detection sensitivity and separation resolution provided by thermal gel electrophoresis enabled rapid screening of native protein variants.