Enrichment of Carbonylated Proteins Using a Surface-Modified Microfluidic Platform (original) (raw)

Abstract

Protein oxidation occurs as a result of exposure to oxidative stress. Protein oxidation has been implicated in aging and many age-related disease states. Protein oxidation is a post-translational modification (PTM) to proteins and can occur in over 35 ways. One form of protein oxidation is carbonylation, an irreversible PTM of proteins where an aldehyde bonds to an amino acid residue, most commonly lysine, proline, theorenine, and arginine. These proteins are used as common markers for oxidative stress within a system. We report a proof of principle study for the use of oxalyldihydrazide as a crosslinker for enrichment of carbonylated proteins within a microfluidic chip. Surface modification steps are characterized and analyzed using analytical techniques. Surface elemental analysis and roughness were examined by X-ray photon spectroscopy (XPS) and atomic force spectroscopy (AFM), respectively. Additionally, fluorescence microscopy was utilized for chemical functional groups mapping to verify the presence of desired crosslinkers. We use in vitro oxidized cytochrome c as a model protein for testing specificity of the methodology for carbonylated proteins. We then mix oxidized cytochrome c with TRITC-BSA to determine selectivity of the method. Capture and elution of the proteins are determined quantitatively. The method demonstrates the potential of using microfluidic platforms for the enrichment of carbonylated proteins.

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