Biomedical subjects

E Bonneil

Publications and source records attributed to E Bonneil.

3 recordsLinked to original sources

PubMed1999

Characterization of a solid-phase extraction device for discontinuous on-line preconcentration in capillary electrophoresis-based peptide mapping.

Peptide mapping by capillary electrophoresis (CE) with UV detection is problematic for the characterization of proteins that can only be obtained at low micromolar concentrations. Dilution of peptide fragments during digestion of the protein can further reduce the detection sensitivity in peptide mapping to the point where analysis at sub-micromolar concentrations is not possible. A remedy to this problem is preconcentration (sample enrichment) of the proteolytic digest by solid-phase extraction (SPE). To minimize non-specific adsorptive losses during sample handling, on-line SPE-CE is preferred. However, packed-inlet SPE-CE is not always feasible due to either instrument or sample limitations. We describe here a simple method of preconcentration by discontinuous on-line SPE-CE, specifically applied to peptide mapping in low-pH separation buffer after protein digestion in a solid-phase enzyme microreactor. The SPE-CE system does not require application of a low pressure during electrophoretic separation to overcome reversed electroosmotic flow because the preconcentrator device is disconnected from the separation capillary before the electric field is applied. Up to a 500-fold preconcentration factor can be achieved with this device, which can be reused for many samples. Parameters such as the volume of desorption solution, the adsorption/desorption (chromatographic) process, reproducibility of packing the SPE preconcentrator and effects of sample concentration on the peptide map are investigated.

Adsorption↗

PubMed1995

Acyclic oligonucleotide analogues.

Acyclic analogues of oligothymidylate and oligoadenylate and their alternating copolymers were synthesized to study their thermal melting, their stability against snake venom phosphodiesterase and their primer/template properties using the Klenow fragment of the Escherichia coli DNA polymerase I enzyme. Acyclic dodecaadenylate (GlyA)12 hybridized to dodecathymidylate p(dT)12, and the complex presented a sharp melting with a Tm at 24 degrees C. This association was confirmed by circular dichroism curves which were similar to those of the natural oligonucleotide duplexes in A-conformation. (GlyA)12 proved very stable against snake venom phosphodiesterase hydrolysis. The reaction rate was more than 10,000 times slower than that of p(dT)12. (GlyA)12 served as a primer for the Klenow DNA polymerase. When (GlyA)12 was complexed with the poly(dT) template, the enzyme polymerized dATP but the reaction was much slower than with the (GlyT)12 primer. Molecular modelling of atactic (GlyA)12.(dT)12 of the A-conformation indicates that this conformation is energetically possible.

Adenine↗

PubMedDate not supplied

On-line solid-phase preconcentration for sensitivity enhancement in capillary electrophoresis.

Capillary electrophoresis is attractive for the analysis of biological samples because only a few nanoliters of sample need to be injected. Indeed, optimal resolution is achieved when the injected volume is 1% or less of the total capillary volume. Unfortunately, this advantage leads to severe detection limitations compounded by the fact that many analytes in a biological sample are present at very low concentrations. To overcome the detection sensitivity limitations of CE, nonspecific, on-line preconcentration has been employed in a variety of applications. This technique is based on inserting a small quantity of reversed-phase material (e.g., C18 particles or membrane) near the inlet of the CE capillary for sample enrichment by solid-phase extraction. A detachable cartridge containing the solid phase is relatively simple to construct and permits the injection of large sample volumes (1-100 microL) into the capillary. Elution of adsorbed analyte in less than 100 nL of solvent permits a 100-1000 fold improvement in the effective concentration limit of detection, depending on the hydrophobicity of the compound. Detection of analytes present in complex mixtures at concentrations as low as 20 amol/microL has been reported when using an on-line preconcentration device in conjunction with CE-mass spectrometry. In this review, the potential merits of the technique are described and some examples of direct analysis of biological samples without rigorous off-line pretreatment are given.

Animals↗