A weak cation-exchange monolith as stationary phase for the separation of peptide diastereomers by CEC.

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/ß-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.

Separation of peptide diastereomers using CEC and a hydrophobic monolithic column.

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.

9-Fluorenylmethoxycarbonyl-labeled peptides as substrates in a capillary electrophoresis-based assay for sirtuin enzymes.

Sirtuins are the class III histone deacetylases that catalyze the deacetylation of acetyl-lysine residues of histones and other proteins using nicotinamide adenine dinucleotide (NAD(+)) as the cofactor. The reaction yields the deacetylated protein, nicotinamide, and 2'-O-acetyl-ADP-ribose. Three 9-fluorenylmethoxycarbonyl (Fmoc)-labeled peptides derived from the amino acid sequence of p53, Fmoc-KK(Ac)-NH(2), Fmoc-KK(Ac)L-NH(2), and Fmoc-RHKK(Ac)-NH(2), were characterized as substrates for two of the human sirtuins: SIRT1 and SIRT2. The deacetylation was monitored by a validated capillary electrophoresis assay. Efficient deacetylation by SIRT1 and SIRT2 was demonstrated for all three peptide substrates. The kinetics of the enzymatic reaction was determined with the Michaelis constants (K(m)) varying between 16.7 and 34.6 microM for SIRT1 and between 34.7 and 58.6 microM for SIRT2. Resveratrol did not function as an activator for SIRT1 using the Fmoc-labeled peptides as SIRT substrates. The IC(50) values of sirtinol using the three peptide substrates were determined. Further sirtuin inhibitors were also evaluated.

Capillary electrophoresis-based sirtuin assay using non-peptide substrates.

Sirtuins are NAD(+)-dependent class III histone deacetylases, which catalyze the deacetylation of acetyl-lysine residues of histones and other protein substrates yielding the deacetylated protein, nicotinamide and 2'-O-acetyl-ADP-ribose. Two lysine amide derivatives containing dansyl (Dns) or 7-dimethylaminocoumarin (DMAC) residues, i.e. Dns-K(Ac)-NH(2) and DMAC-K(Ac)-NH(2), were synthesized and evaluated as substrates for human sirtuin 1. A CZE method with field amplified sample injection and a MEKC method with sweeping were established and validated for monitoring the deacetylation process of Dns-K(Ac)-NH(2) and DMAC-K(Ac)-NH(2), respectively. Deacetylation by sirtuin 1 was demonstrated for both of the substrates. The Michaelis-Menten constants, K(m), were 88.0µM for Dns-K(Ac)-NH(2) and 42.9µM for DMAC-K(Ac)-NH(2). The applicability of the methods was demonstrated using known sirtuin inhibitors. Resveratrol did not activate sirtuin 1 using the present CE-based enzyme assay. The results indicated that the lysine derivatives can be used in sirtuin assays instead of peptide substrates.

Application of phase optimized liquid chromatography to oligopeptide separations.

Stationary phase optimized liquid chromatography (POPLC) has been applied to the separation of oligopeptides. The retention factors and theoretical plate numbers of 13 peptides were determined on five different stationary phases. Based on these values, an optimal stationary phase composition of 250mm total length consisting of 3 segments of 20mm octadecyl silica, 10mm phenyl silica and 220mm embedded polar octadecyl silica was calculated by the optimizer software. Good agreement between the calculated and experimental chromatograms was observed. In order to achieve short analysis time and baseline separation of all peptides gradient elution and optimization of the column temperature were performed. The optimized mobile phase conditions consisted of (A) acetonitrile and (B) 0.025M aqueous sodium phosphate buffer, pH 3.0, operated at 10% A (0-12min) followed by 10-50% A (12-32min) at a flow rate of 0.5mLmin(-1) and column temperature of 35 degrees C. Using these conditions all peptides could be separated in less than 30min with good resolution and peak symmetry.

Development of a capillary electrophoresis-based assay of sirtuin enzymes.

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.