Miniaturized analytical systems for mass spectrometry-based protein studies

Current proteomic strategies depend strongly on the development of analytical methodologies and instrumentation. In parallel to the development of mass spectrometry (MS) - based proteomic workflows, microfluidic devices emerged in this field as a flexible tool for rapid and sensitive protein studies. In this context, the present work focuses on the development of miniaturized analytical systems for protein studies, especially by electrospray ionization mass spectrometric detection. Several approaches have been proposed to complement the mass spectrometric analysis of tryptic peptides using chemical tagging to isolate specific subclasses of proteins by affinity baits or for quantitation purposes. Optimization of a chemical tagging reaction in a sandwich mixer-reactor that consists of mixing two solutions providing the reactants in a channel, one central laminar flow being sandwiched between two outer flow solutions, was first investigated by finite-element method. The numerical simulations have highlighted the importance of positioning the reactant presenting the lowest diffusion coefficient close to the sidewall, as well as decreasing the outer flow velocities, to enhance a chemical reaction because of the parabolic flow profile across the microchannel. This optimization is even more relevant for consecutive reactions. As infusion-based electrospray microchips are known to present great advantages in terms of sensitivity compared to standard ionization source, an ESI emitter microchip based on polymer photo-ablation was designed. First, the hyphenation of the chip in a LC-MS workflow was successfully achieved and evaluated by comparison with a standard pneumatically-assisted ESI source. To perform enhanced on-chip post-column derivatization of peptides, a substitute design to the sandwich mixer-reactor was explored to improve reaction in a microchip before ESI – MS analysis. The electrospray micromixer chip includes a passive mixing unit to perturbate the flow along the microchannel. The mixing efficiency was demonstrated by fluorescence imaging, and on-chip chemical derivatization of peptides and kinetic studies before mass spectrometry were achieved. The on-line LC-MS derivatization of cysteinyl peptides was shown to provide a more confident and accurate protein identification by adding information on the peptidic sequence. Within the development of electrospray microchips for mass spectrometry, many efforts have been put forward in the hyphenation of functional units. Immobilization of functionalized stationary phase in microfluidic systems is widely used to achieve protein or peptide isolation, sample cleaning, separation or reaction. In this context, magnetic beads have proven to be quite useful compared to other methods, such as immobilized packed beads or monoliths, because of their high and reversible magnetization, and various surface functionalization. A polymer microchip including a magnetic track array was designed to focus the magnetic field generated by permanent magnets towards precise locations along the microchannel. A multi-plug magnetic bead capture was obtained and a significant increase of bead capture efficiency was demonstrated both numerically and experimentally, which is beneficial for affinity separation applications, as example. The research in microfluidic front-end devices for mass spectrometry is actually oriented to the development of multi-spray interfaces for high-throughput analysis. An electrospray microchip with a multi-track array was developed. The capability of the device to screen alternatively up to six samples and to perform relative quantification was assessed. Soft ionization techniques have revolutionized the analysis of proteins by mass spectrometry. In contrast with the major content of the thesis dealing with microfluidic systems for protein analysis, the last chapter presents a novel ambient ionization method, so-called membrane-desorption electrospray ionization (M-DESI). The principle is similar to the desorption electrospray ionization (DESI) method. But, in the M-DESI approach the sample is desorbed directly from a mesh membrane positioned vertically and in-axis between the ESI emitter and the mass spectrometer inlet. Analysis of peptides and proteins was demonstrated as a proof-of-concept, offering great perspectives for detection after separation on membrane

Magnetic track array for efficient bead capture in microchannels

Magnetism-based microsystems, as those dedicated to immunoaffinity separations or (bio)chemical reactions, take benefit of the large surface area-to-volume ratio provided by the immobilized magnetic beads, thus increasing the sensitivity of the analysis. As the sensitivity is directly linked to the efficiency of the magnetic bead capture, this paper presents a simple method to enhance the capture in a microchannel. Considering a microchannel surrounded by two rectangular permanent magnets of different length (L m = 2, 5, 10mm) placed in attraction, it is shown that the amount of trapped beads is limited by the magnetic forces mainly located at the magnet edges. To overcome this limitation, a polyethylene terephthalate (PET) microchip with an integrated magnetic track array has been prototyped by laser photo-ablation. The magnetic force is therefore distributed all along the magnet length. It results in a multi-plug bead capture, observed by microscope imaging, with a magnetic force value locally enhanced. The relative amount of beads, and so the specific binding surface for further immunoassays, presents a significant increase of 300% for the largest magnets. The influence of the track geometry and relative permeability on the magnetic force was studied by numerical simulations, for the microchip operating with 2-mm-long magnet

Electrochemical reactions and ionization processes

Electrochemical or photo-electrochemical reactions in both electrospray ionization and laser desorption ionization are discussed stressing the role of the electrode reaction in influencing the ionization process. In particular, upon application of a high voltage during electrospray ionization, the emitter includes a working electrode, where redox reactions are observed, such as electro-generation of benzoquinone and metal ions. In contrast, the target plate in laser-induced desorption ionization also acts as a photo-electrode, especially when modified with a mesoporous semiconductor. We illustrate here how these electrochemical reactions can be used for tagging purposes, and for oxidative or reductive dissociation reactions

Antioxidant power measurement in platelet concentrates treated by two pathogen inactivation systems in different blood centres.

The antioxidant power measurement can be useful to validate the execution of the pathogen inactivation treatment of platelet concentrates. The aim of this study is to evaluate the technology on different blood preparations including INTERCEPT and Mirasol treatments that are in routine use in Belgium and Luxemburg. The antioxidant power measurement was tested on 78 apheresis platelet concentrates and 54 pools of buffy-coats-derived platelet concentrates before and after INTERCEPT treatment. In addition, 100 Reveos platelet pools were tested before and after Mirasol treatment. The antioxidant power was quantified electrochemically using disposable devices and was expressed as equivalent ascorbic acid concentration. Mean results for apheresis platelet concentrates were of 90 ± 14 and 35 ± 10 µmol/l eq. ascorbic acid before and after INTERCEPT treatment, respectively. The mean results for pools of buffy-coats-derived platelet concentrates were of 81 ± 10 and 29 ± 4 eq. µmol/l ascorbic acid before and after INTERCEPT treatment, respectively. For buffy-coats-derived platelet concentrates treated by Mirasol technology, the mean results were of 98 ± 11 and 32 ± 10 µmol/l eq. ascorbic acid before and after illumination, respectively. The antioxidant power significantly decreases with pathogen inactivation treatments for platelet concentrates treated by INTERCEPT or Mirasol technologies

Transfusion-transmitted cytomegalovirus: behaviour of cell-free virus during blood component processing. A study on the safety of labile blood components in Switzerland.

BACKGROUND Nowadays, most blood products are leukocyte-reduced. After this procedure, the residual risk for transfusion transmitted cytomegalovirus (TT-CMV) is mostly attributed to cell-free viruses in the plasma of blood donors following primary infection or viral reactivation. Here, objectives are: 1) to study the behaviour of cell-free CMV through the blood component processing; 2) to determine the anti-CMV seroprevalence, the level of viremia, the window-period in blood donor population; and 3) to identify cases of TT-CMV in bone marrow transplant (BMT) recipients. MATERIALS AND METHODS Cell-free CMV was injected into blood bags originating from regular donors. Blood components were processed according to either the CompoSelect® or the CompoFlow® (Fresenius Kabi AG) techniques. Samples were analysed at each step for presence of virus DNA using quantitative polymerase chain reaction (PCR). The anti-CMV seroprevalence in our donor population was taken from our donor data system. The viremia was assessed in pooled plasmas samples from routine donations by quantitative PCR. Medical charts of 165 BMT anti-CMV seronegative recipients/anti-CMV seronegative donors who received CMV-unscreened blood products were reviewed. RESULTS Cell-free CMV passes without any decrease in viral load through all stages of blood processing. The anti-CMV seroprevalence was 46.13%. Four DNA positive samples out of 42,240 individual blood donations were identified (0.009%); all had low levels of viremia (range 11-255 IU/mL). No window-period donation was identified. No TT-CMV was found. DISCUSSION Cell-free CMV remains a concern with current blood component processing as it passes through all the processes. However, since low levels of CMV DNA were identified in the donations tested, and no BMT recipients had TT-CMV, the residual threat of TT-CMV after leukocyte reduction appears to be very low