Jean-Pierre Chervet

Instrumental requirements for nanoscale liquid chromatography.

Nanoscale liquid chromatography (nano-LC), with packed columns of typically 75 μm i.d. × 15 cm length, packed with C18, 5 μm of stationary phase, and optimal flow rates of 180 nL/min, can be considered as a miniaturized version of conventional HPLC. Using the down-scaling factor, which corresponds to the ratio of the column diameter in square, (d(conv)/d(micro))(2), excellent agreement between the theoretically calculated values and the values obtained using the down-scaling factor (∼3800) has been observed. This factor was applied to all system components, including flow rate, injection and detection volumes, and connecting capillaries. Down-scaling of a conventional HPLC system to a nano-LC system is easy to realize in practice and involves using a microflow processor for nanoflow delivery (50-500 nL/min), a longitudinal nanoflow cell (≤3 nL), a microinjection valve (≤ 20 nL), low-dispersion connecting tubing, and zero dead volume connections. Excellent retention time reproducibility was measured with RSD values of ±0.1% for isocratic and ±0.2% for gradient elution. Plates counts of more than 100 000/m indicate the excellent performance of the entire nano-LC system. With minimal detectable amounts of proteins in the low femtomole and subfemtomole ranges (e.g., 520 amol for bovine serum albumin), high mass sensitivity was found, making nano-LC attractive for the microcharacterization of valuable and/or minute proteinaceous samples. Coupling nano-LC with concomitant mass spectrometry using nanoscale ion spray or electrospray interfaces looks very promising and is obviously the next step for future work.

Z-shaped flow cell for UV detection in capillary electrophoresis

Abstract A longtitudinal (Z-shaped) flow cell for improved UV detection for capillary electrophoresis was examined and compared with perpendicular (on-column) detection. Bending of a small section of the capillary column into a Z-shaped flow cell has no adverse influence on the electrophoresis process. With enhancements in signal-to-noise ratio of up to 6-fold for 3-mm Z-shaped flow cells, a considerable improvement in detectability is possible. The increase in signal of up to 14-fold in comparison with on-column UV detection illustrates the potential of this new type of capillary flow cell. the loss in resolution caused by the extnded path length is less pronounce than expected, and can be tolerated in many of the CE separation modes.

Optimised injection techniques for micro and capillary liquid chromatography

Abstract Large volume injections —involving on-column focusing— were evaluated for packed micro and capillary liquid chromatography columns of 300 μm and 1.0 mm inner diameter (I.D.) respectively. It was found that the I.D. of the injection loop plays a critical role in sample dispersion, effecting peak asymmetry and injection reproductibility. The use of injection loops with too large or too small an I.D. resulted in a reduced injection performance. Ideally the I.D. should be in between 100–150 μm. Further it was investigated what influence the loop volume and the use of a low dispersion injection technique had on the column stability. The combination of large volume injections—up to 1 μl and 5 μl for 300 μm and 1.0 mm I.D. columns, respectively—and additional switching of the injection valve did not affect column lifetime. Typical decreases of only 10% in efficiency over 1500 injections were found.

A novel electrochemical method for efficient reduction of disulfide bonds in peptides and proteins prior to MS detection

AbstractA novel electrochemical (EC) method for fast and efficient reduction of the disulfide bonds in proteins and peptides is presented. The method does not use any chemical agents and is purely instrumental. To demonstrate the performance of the EC reactor cell online with electrospray mass spectrometry, insulin and somatostatin were used as model compounds. Efficient reduction is achieved in continuous infusion mode using an EC reactor cell with a titanium-based working electrode. Under optimized conditions, the presented method shows almost complete reduction of insulin and somatostatin. The method does not require any special sample preparation, and the EC reactor cell makes it suitable for automation. Online EC reduction followed by collision-induced dissociation fragmentation of somatostatin showed more backbone cleavages and improved sequence coverage. By adjusting the settings, the EC reaction efficiency was gradually changed from partial to full disulfide bonds reduction in α-lactalbumin, and the expected shift in charge state distribution has been demonstrated. The reduction can be controlled by adjusting the square-wave pulse, flow rate or mobile phase composition. We have shown the successful use of an EC reactor cell for fast and efficient reduction of disulfide bonds for online mass spectrometry of proteins and peptides. The possibility of online and gradual disulfide bond reduction adds a unique dimension to characterization of disulfide bonds in mid- and top-down proteomics applications. FigurePrinciple of electrochemical reduction of disulfide bonds in proteins

Electrochemical simulation of oxidation processes involving nucleic acids monitored with electrospray ionization–mass spectrometry

AbstractOxidation is commonly involved in the alteration of nucleic acids giving rise to diverse effects including mutation, cell death, malignancy, and aging. We demonstrate that electrochemistry represents an efficient and fast method to mimic oxidative modification of nucleic acids occurring in biological systems. Oxidation reactions were performed in a thin-layer cell employing a conductive diamond electrode as the working electrode and were monitored with electrospray ionization–mass spectrometry. Mass voltammograms were acquired for guanosine, adenosine, cytidine, and uridine. The observed oxidation potentials increased in the order guanosine<<adenosine<cytidine<uridine. Oxidation products of guanosine were characterized using high-resolution (tandem) mass spectrometry performed with a quadrupole–quadrupole time-of-flight instrument. On the basis of these experiments, it was concluded that the initial electrode reaction involves a one-electron, one-proton step to give a free radical. The primary oxidation product represents the starting point for a number of follow-up reactions, including guanosine dimerization as well as further oxidation to 8-hydroxyguanosine. Similar results were obtained for guanosine monophosphate and the corresponding dinucleotide. Furthermore, the guanosine radical was identified as an important intermediate for the formation of a covalent adduct with acetaminophen. This observation sheds new light on the mechanism of adduct formation as it demonstrates that oxidative activation of both the nucleobase and the adduct-forming agent is necessary to observe a detectable amount of adduct species. FigureOn-line electrochemistry/mass spectrometry is fast, simple, and convenient method to study the impact of oxidation reactions on different kinds of nucleic acid species, including nucleosides, nucleotides and small oligomers. The initial electrode reaction involves a 1e−, 1H+ step to give a free radical. The primary oxidation product represents the starting point for a number of follow-up reactions.

Sodium Dodecyl Sulphate Removal from Tryptic Digest Samples for On-line Capillary Liquid Chromatography/Electrospray Mass Spectrometry

An on-line microcolumn switching method was developed for the removal of sodium dodecyl sulphate (SDS) from tryptic digest samples. The system includes two micro-precolumns: a specific ionic detergent trapping column and a preconcentration column. Characterization of the proteinaceous samples, after isolation from the SDS, was performed by capillary liquid chromatography (LC) with UV absorption detection and electrospay mass spectrometry (ESI-MS). Loading and clean-up of the samples and regeneration of the detergent trapping column were performed at 50 microl min(-1), resulting in sample clean-up times of only 30 s. SDS-containing tryptic digested protein samples were directly applied to the micro-precolumns without any previous sample pretreatment. The developed microcolumn switching method permits the on-line analysis of small tryptic digest samples by capillary LC/ESI-MS in the presence of SDS. The method is completely automated and can be performed unattended. The maximum amount of SDS, in terms of loadability and breakthrough, were determined. Also studied were the selection of the loading and clean-up solvents and the recovery of the peptides. Chromatographic separations and mass spectral data confirmed the removal of SDS.

Ascorbic acid for homogenous redox buffering in electrospray ionization-mass spectrometry

AbstractElectrospray ionization (ESI) involves the dispersion of a liquid containing analytes of interest into a fine aerosol by applying a high potential difference to the sample solution with respect to a counter electrode. Thus, from the electrochemical point of view, the ESI source represents a two-electrode controlled-current electrochemical flow cell. The electroactive compounds part of the solvent sprayed may be altered by occurring electrolysis (oxidation in positive ion mode and reduction in negative ion mode). These reactions can be troublesome in the context of unknown identification and quantification. In the search for a simple, inexpensive, and efficient way to suppress electrochemical oxidation in positive ESI, the usability of ascorbic acid, hydroquinone, and glutathione for homogenous redox buffering was tested. Performance of the antioxidants was assessed by analyzing pharmaceutical compounds covering a broad range of functional groups prone to oxidation. Different emitter setups were applied for continuous infusion, flow injection, and liquid chromatography/mass spectrometry experiments. Best performance was obtained with ascorbic acid. In comparison to hydroquinone and glutathione, ascorbic acid offered superior antioxidant activity, a relatively inert oxidation product, and hardly any negative effect on the ionization efficiency of analytes. Furthermore, ascorbic acid suppressed the formation of sodiated forms and was able to induce charge state reduction. Only in the very special case of analyzing a compound isobaric to ascorbic acid, interference with the low-abundant [ascorbic acid+H]+ signal may become a point of attention. FigureAscorbic acid efficiently suppresses analyte oxidation and formation of sodiated forms in positive electrospray ionization

Automated on-line ionic detergent removal from minute protein/peptide samples prior to liquid chromatography-electrospray mass spectrometry

An automated on-line ionic detergent removal pre-column system coupled to capillary liquid chromatography-electrospray mass spectrometry is described. The system involves two micro precolumns, composed of a specific ionic detergent trapping column and a preconcentration column, respectively, and a packed 300 microns I.D. analytical column. Sample loading to the micro precolumns and regeneration of the detergent trapping column were performed at a flow-rate of 50 microliters/min, while the flow-rate through the analytical column was set at 5.0 microliters/min. Ionic detergent-containing tryptic-digested protein samples were directly applied to the micro precolumns without sample pretreatment and were analysed by UV absorption detection and electrospray mass spectrometry. The presented system allows for the fully automated removal of SDS with virtually no loss in protein/peptides. Maximum SDS load and breakthrough have been determined. Excellent protein recovery and complete removal of SDS is found. The chromatographic separation after SDS removal was completely restored and equalled the reference chromatogram. Mass spectral data confirm these findings. Finally, this technique allows for SDS removal from minute protein samples without the need for any sample handling.

Size-exclusion chromatography performed in capillaries: Studies by liquid chromatography–mass spectrometry

Miniaturization of the chromatographic column led to increased sensitivity and shortened time of analysis. In our work we applied 300 microm I.D. capillaries packed with a novel stationary phase Superdex Peptide for the size-exclusion chromatography, capable of separating molecules within the mass range of 0.1-7 kDa. Here we proved that such capillary columns can operate effectively at high sensitivity. Several peptide mixtures were efficiently chromatographed and analyzed on line with electrospray ionization mass spectrometry as a detection technique. A CNBr peptide map, derived from human globin alpha subunit, was effectively separated using this method. These fragments are difficult to elute from the reversed-phase column at low pH, therefore, such approach can be considered as a complementary to other separation techniques, in particular for analyzing hydrophobic components and complex mixtures.

Studying the reducing potencies of antioxidants with the electrochemistry inherently present in electrospray ionization-mass spectrometry

AbstractIn this proof-of-principle study, the applicability of electrospray ionization-mass spectrometry (ESI-MS) to characterize the reducing potencies of natural antioxidants is demonstrated. The ESI source represents a controlled-current electrochemical cell. The interfacial potential at the emitter electrode will be at or near the electrochemical potential of those reactions that sufficiently supply all the required current for the ESI circuit. Indicator molecules prone to oxidation in ESI such as amodiaquine were used to visualize the impact of reducing compounds on the interfacial potential. The extent of inhibition of the oxidation of the indicator molecule was found to be dependent on the kind and amount of antioxidant added. Concentration–inhibition curves were constructed and used to compare reducing potencies and to rank antioxidants. This ranking was found to be dependent on the electrode material–indicator molecule combination applied. For fast and automated characterization of the reducing potencies of electrochemically active molecules, a flow-injection system was combined with ESI-MS. Liquid chromatography was used to process complex biological samples, such as red and white wine. Due to their high content of different polyphenols, red wine fractions were found to exhibit higher reducing potencies than the corresponding white wine fractions. Furthermore, for 14 important natural antioxidants, the results obtained with the controlled-current EC–ESI-MS assay were compared to those obtained with chemical antioxidant assays. Irrespectively of the kind of assay used to test the reducing potency, gallic acid, quercetin, and epicatechin were found to be potent reductants. Other antioxidants performed well in one particular assay only. This observation suggests that different kinds of redox and antioxidant chemistry were assessed with each of the assays applied. Therefore, several assays should be used to comprehensively study antioxidants and their reducing potencies. FigureFractions of a red wine sample were screened by ESI-MS for compounds showing reducing potency.