Abstract
Fluorous-modified surfaces have emerged as a powerful tool for the immobilization of fluorous-tagged biomolecules based on their specificity and the strength of fluorous-fluorous interactions. To fabricate a fluorous-based protein microarray, we designed two strategies for site-specific modification of proteins with a fluorous tag: attaching the fluorous tag to the C-termini of expressed proteins by native chemical ligation (NCL) or to the Fc domain of antibodies through boronic acid (BA)-diol interactions. The perfluoro-tagged proteins could be easily purified by fluorous-functionalized magnetic nanoparticles (MNPs) and immobilized on a fluorous chip with minimal non-specific adsorption. Importantly, proteins immobilized on the solid support through non-covalent fluorous-fluorous interactions were sufficiently stable to withstand continuous washing. We believe that this fluorous-fluorous immobilization strategy will be a highly valuable tool in protein microarray fabrication.
Highlights
One of the most powerful tools in proteomics is the protein microarray, which utilizes immobilized proteins on a solid support for high-throughput analysis of biochemical properties and biological activities[1]
To access BA-containing probe, Boc-protected cysteic acid linker (11) was incorporated on amine form of 8 to afford an intermediate 12 (68% over two steps), which was followed by Boc deprotecion and subsequent 1-ethyl-3-(3-dimethylaminorpopyl)carbodiimide hydrochloride (EDC-HCl)/1-hydroxybenzotriazole (HOBT)mediated coupling with 3-carboxyphenylboronic acid to afford fluorous-tagged BA probe 2 in a modest 18% yield over two-steps
For the synthesis of arginine-containing probe, Fmoc-Arg(Pbf)-OH was first reacted with the amine generated from flourous tag 8, and extension of the synthesis of Fmoc-protected intermediate was carried out using 20% piperidine in DMF as the Fmoc deprotecting agent followed by coupling to the carboxylic acid of the incoming amino acid Boc-Cys(Trt)-OH affording 13 (53%, 4-steps)
Summary
One of the most powerful tools in proteomics is the protein microarray, which utilizes immobilized proteins on a solid support for high-throughput analysis of biochemical properties and biological activities[1]. Popular examples include DNA-directed immobilization[12], avidin-biotin interaction[13], and affinity immobilization of His-tagged fusion proteins to a metal-chelate support[14, 15] Such methods have potential drawbacks like the non-specific adsorption of non-target proteins in complex biological samples to the solid support, and insufficient strength of non-covalent interactions, which make the immobilized proteins susceptible to falling off after prolonged incubation under complex biological conditions (such as serum), or continuous vigorous washing. To take advantage of the high specificity of noncovalent binding strategies, the immobilization surface must be able to resist the adsorption of other proteins that lack an affinity tag. We developed a straightforward protein microarray fabrication method utilizing a fluorous-based surface for the oriented and noncovalent immobilization of proteins on the surface through fluorous-fluorous interactions. Proteins could be modified with the fluorous probes by either native chemical ligation (NCL) (for expressed proteins, Fig. 1a) or boronate formation (for antibodies, Fig. 1b) and immobilized on fluorous surfaces via fluorous interactions with minimal background
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