Biomedical subjects

Laurent Rieux

Publications and source records attributed to Laurent Rieux.

3 recordsLinked to original sources

PubMed2006

Fast, high-efficiency peptide separations on a 50-microm reversed-phase silica monolith in a nanoLC-MS set-up.

Proteomic studies have stimulated the development of novel stationary phases in miniaturised chromatographic columns that permit high linear flow velocities and exhibit high resolving power. In this work, a 50-microm reversed-phase silica-based monolith was chromatographically characterised for its use in proteomics applications using a nanoLC-MS set-up. It showed high efficiency for the separation of tryptic peptides under isocratic elution conditions (HETP(min)=5-10 microm at 2.4 mm/s). Flow rates up to 1.95 microL/min (18.4 mm/s) and gradient slopes up to an unusually fast 9% could be used. This resulted in rapid separations of peptide mixtures, with peak widths at half height of between 5 and 10 s. The 50-microm monolithic column was used to analyse depleted serum from a cervical cancer patient at a throughput of one sample per 30 min.

Amino Acid Sequence↗

PubMed2005

Silica monolithic columns: synthesis, characterisation and applications to the analysis of biological molecules.

In recent years, proteomics has been a subject of intense research. The complexity of proteomics samples has fostered technological developments. One of these addresses the need for more efficient and faster separations. Monolithic columns prepared from organic and silica monomers offer very efficient separations at low back-pressure. Silica-based monoliths have small-sized skeletons and a bimodal pore size distribution with microm-sized throughpores and nm-sized mesopores. This gives silica-based monoliths favourable properties for high-efficiency, fast separations, like a low-pressure drop across the column, fast mass transfer kinetics and a high binding capacity.

Chemistry, Organic↗

PubMedDate not supplied

Chemically modified, immobilized trypsin reactor with improved digestion efficiency.

Tryptic digestion followed by identification using mass spectrometry is an important step in many proteomic studies. Here, we describe the preparation of immobilized, acetylated trypsin for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was obtained with two types of modified-trypsin beads with a contact time of only 4 s, while corresponding unmodified-trypsin beads gave only incomplete digestion. The digestion rate of myoglobin, a protein known to be rather resistant to proteolysis, was not altered by acetylating trypsin and required a buffer containing 35% acetonitrile to obtain complete digestion. The use of acetylated-trypsin beads led to fewer interfering tryptic autolysis products, indicating an increased stability of this modified enzyme. Importantly, the modification did not affect trypsin's substrate specificity, as the peptide map of myoglobin was not altered upon acetylation of immobilized trypsin. Kinetic digestion experiments in solution with low-molecular-weight substrates and cytochrome c confirmed the increased catalytic efficiency (lower K(M) and higher k(cat)) and increased resistance to autolysis of trypsin upon acetylation. Enhancement of catalytic efficiency was correlated with the number of acetylations per molecule. The favorable properties of the new chemically modified trypsin reactor should make it a valuable tool in automated protein analysis systems.

Acetonitriles↗