An integrated on-chip sirtuin assay.
A microchip-based assay to monitor the conversion of peptide substrates by human recombinant sirtuin 1 (hSIRT1) is presented. For this purpose a fused silica microchip consisting of a microfluidic separation structure with an integrated serpentine micromixer has been used. As substrate for the assay, we used a 9-fluorenylmethoxycarbonyl (Fmoc)-labeled tetrapeptide derived from the amino acid sequence of p53, a known substrate of hSIRT1. The Fmoc group at the N-terminus resulting from solid-phase peptide synthesis enabled deep UV laser-induced fluorescence detection with excitation at 266 nm. The enzymatic reaction of 0.1 U/μL hSIRT1 was carried out within the serpentine micromixer using a 400 μM solution of the peptide in buffer. In order to reduce protein adsorption, the reaction channel was dynamically coated with hydroxypropylmethyl cellulose. The substrate and the deacetylated product were separated by microchip electrophoresis on the same chip. The approach was successfully utilized to screen various SIRT inhibitors.
Institute of University of Leipzig, Analytical Chemistry, Leipzig, Germany.
Stefan
S
Ohla
Reinhild
R
Beyreiss
Gerhard K E
GK
Scriba
Yi
Y
Fan
Detlev
D
Belder
D046209:Electrophoresis, Microchip / Q000295:instrumentation* / Q000379:methods*; D004867:Equipment Design; D005449:Fluorenes / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D065347:Hypromellose Derivatives; D008747:Methylcellulose / Q000031:analogs & derivatives / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D056564:Sirtuin 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C496789:9-fluorenylmethoxycarbonyl; D005449:Fluorenes; D011994:Recombinant Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D065347:Hypromellose Derivatives; D008747:Methylcellulose; C447939:SIRT1 protein, human; D056564:Sirtuin 1