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Sirtuins are a family of nicotinamide adenine dinucleotide (NAD(+))-dependent enzymes catalyzing the deacetylation of acetyl-lysine residues of histones and other proteins. Three 9-fluorenylmethoxycarbonyl (Fmoc)-labeled peptide substrates derived from the amino acid sequence of p53, i.e. Fmoc-KK(Ac)-NH(2), Fmoc-KK(Ac)L-NH(2) and Fmoc-RHKK(Ac)-NH(2), were synthesized and evaluated as substrates of the human isoenzyme SIRT1. The acetylated and respective deacetylated peptides as well as nicotinamide as the reaction product of nicotinamide adenine dinucleotide were separated by capillary electrophoresis in a fused-silica capillary using 200 mM phosphate-Tris buffer, pH 2.7. Sodium hydroxide-mediated sample stacking was performed in order to overcome peak asymmetry due to the high salt and acid content of the sample as well as to enhance UV detection sensitivity. The assay was subsequently validated. Upon incubation of the acetylated peptides for 60 min in the presence of 2.5 U of SIRT1 at least 87% of the peptides was deacetylated, indicating that the new derivatives are efficient substrates of the enzyme.

A neutral hydrophobic monolith prepared by radical in situ copolymerization of lauryl methacrylate and ethylene dimethacylate has been evaluated for the CEC separation of diastereomers of small peptides using acidic mobile phases containing ACN as organic modifier. Using an acidic mobile phase, the peptides migrated due to their own electrophoretic mobility. Hydrophobic interactions with the stationary phase contributed to the separation. Peptide mobility and resolution increased with increasing the ACN content. Retention times increased with the pH of the mobile phase. Peak resolution increased with buffer pH and concentration. Di- and tripeptides composed only of L-configured amino acids migrated faster than peptides containing D-amino acids. A mixture of isomeric Asp tripeptides that could not be completely resolved by either CZE or HPLC as well as the 24mer peptides tetracosactide and (16)[D-Lys]-tetracosactide could also be separated by CEC on the hydrophobic monolith.

A CEC weak cation-exchange monolith has been prepared by in situ polymerization of acrylamide, methylenebisacrylamide and 4-acrylamidobutyric acid in a decanol-dimethylsulfoxide mixture as porogen. The columns were evaluated by SEM and characterized with regard to the separation of diastereomers and α/β-isomers of aspartyl peptides. Column preparation was reproducible as evidenced by comparison of the analyte retention times of several columns prepared simultaneously. Analyte separation was achieved using mobile phases consisting of acidic phosphate buffer and ACN. Under these conditions the peptides migrated due to their electrophoretic mobility but the EOF also contributed as driving force as a function of the pH of the mobile phase due to increasing dissociation of the carboxyl groups of the polymer. Raising the pH of the mobile phase also resulted in deprotonation of the peptides reducing analyte mobility. Due to these mechanisms each pair of diastereomeric peptides displayed the highest resolution at a different pH of the buffer component of the mobile phase. Comparing the weak-cation exchange monolith to an RP monolith and a strong cation-exchange monolith different elution order of some peptide diastereomers was observed, clearly illustrating that interactions with the stationary phase contribute to the CEC separations.