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T Cells Recognizing a Peptide Contaminant Undetectable by Mass Spectrometry
Brezar V, Culina S, Osterbye T, Guillonneau F, Chiappetta G, Verdier Y, Vinh J, Wong FS, Buus S, Mallone R.
PLoS One - 6(12) :e28866 - DOI:10.1371/journal.pone.0028866 - 2011
Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted ß-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206-214 epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP206-214 using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP206-214. If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results.
Reversed-phase HPLC and hyphenated analytical strategies for peptidomics
Hesse AM, Ndiaye S, Vinh J.
Methods Mol Biol. - 789 :203-21 - DOI:10.1007/978-1-61779-310-3_13. - 2011
Peptide study and analysis widely involve liquid chromatography. Among the different strategies available, reversed-phase liquid chromatography (RP-HPLC) is one of the methods of choice to separate species in a nontargeted approach. The compounds are sorted according to their hydrophobicity, even though the experimental order of elution could change according to the nature of the mobile phase and the stationary phase. In our work, we have developed protocols to resolve hundred of peptidic species. To overcome the limitations of peak capacity of RP-HPLC alone, it has been coupled downstream to tandem mass spectrometry using two different ionization modes. To overcome the limitations of peak capacity of RP-HPLC MS/MS, it has been coupled upstream to strong cation exchange liquid chromatography. Multidimensional analysis allows for a deeper description of a sample because the limit of detection is often due to a lack of dynamic range of the detection itself rather than due to a lack of sensitivity. In this chapter, different protocols are presented. They should be considered as examples that could be used as starting point for new protocols optimization. Even if RP-HPLC is a universal peptide separation method, it should be optimized according to the specific characteristics of the peptide(s) of interest.
Millifluidic droplet analyser for microbiology
L. Baraban, F. Bertholle, M. Salverda, N. Bremond, P. Panizza, J. Baudry, A.G.M. de Visser, J. Bibette
Lab. Chip - 11(23) :4057-62 - DOI:10.1039/C1LC20545E - 2011
We present a novel millifluidic droplet analyser (MDA) for precisely monitoring the dynamics of microbial populations over multiple generations in numerous (>103) aqueous emulsion droplets (~100 nL). As a first application, we measure the growth rate of a bacterial strain and determine the minimal inhibitory concentration (MIC) for the antibiotic cefotaxime by incubating bacteria in a fine gradient of antibiotic concentrations. The detection of cell activity is based on the automated detection of an epifluorescent signal that allows the monitoring of microbial populations up to a size of ~106 cells. We believe that this device is helpful for the study of population dynamic consequences of microbe-environment interactions and of individual cell differences. Moreover, the fluidic machine may improve clinical tests, as it simplifies, automates and miniaturizes the screening of numerous microbial populations that grow and evolve in compartments with a finely tuned composition.
Force-Velocity Measurements of a Few Growing Actin Filaments
C. Brangbour, O. du Roure, E. Helfer, D. Demoulin, A. Mazurier, M. Fermigier, M.F. Carlier, J. Bibette, J. Baudry
Plos Biology - 9(4) :e10000613 - DOI:10.1371/journal.pbio.1000613 - 2011
The polymerization of actin in filaments generates forces that play a pivotal role in many cellular processes. We introduce a novel technique to determine the force-velocity relation when a few independent anchored filaments grow between magnetic colloidal particles. When a magnetic field is applied, the colloidal particles assemble into chains under controlled loading or spacing. As the filaments elongate, the beads separate, allowing the force-velocity curve to be precisely measured. In the widely accepted Brownian ratchet model, the transduced force is associated with the slowing down of the on-rate polymerization. Unexpectedly, in our experiments, filaments are shown to grow at the same rate as when they are free in solution. However, as they elongate, filaments are more confined in the interspace between beads. Higher repulsive forces result from this higher confinement, which is associated with a lower entropy. In this mechanism, the production of force is not controlled by the polymerization rate, but is a consequence of the restriction of filaments' orientational fluctuations at their attachment point.
Silica sputtering as a novel collective stationary phase deposition for microelectromechanical system gas chromatography column: Feasibility and first separation
J. Vial, D. Thiébaut, F. Marty, P. Guibal, R. Haudebourg, K. Nachef, K. Danaie, B. Bourlon
J. Chrom. A - 1218(21) :3262-66 - DOI:10.1016/j.chroma.2010.12.035 - 2011
Since the late 1970s, approaches have been proposed to replace conventional gas chromatography apparatus with silicon-based microfabricated separation systems. Performances are expected to be improved with miniaturization owing to the reduction of diffusion distances and better thermal management. However, one of the main challenges consists in the collective and reproducible fabrication of efficient microelectromechanical system (MEMS) gas chromatography (GC) columns. Indeed, usual coating processes or classical packing with particulate matters are not compatible with the requirements of collective MEMS production in clean room facilities. A new strategy based on the rerouting of conventional microfabrication techniques and widely used in electronics for metals and dielectrics deposition is presented. The originality lies in the sputtering techniques employed for the deposition of the stationary phase. The potential of these novel sputtered stationary phases is demonstrated with silica sputtering applied to the separation of light hydrocarbons and natural gases. If kinetic characteristics of the sputtered open tubular columns were acceptable with 2500 theoretical plates per meter, the limited retention and resolution of light hydrocarbons led us to consider semipacked sputtered columns with rectangular pillars allowing also significant reduction of typical diffusion distances. In that case separations were greatly improved because retention increased and efficiency was close to 5000 theoretical plates per meter.
Selective extraction of nitroaromatic explosives by using molecularly imprinted silica sorbents
S. Lordel, F. Chapuis-Hugon, V. Eudes, V. Pichon
Anal. Bioanal. Chem - 399(1) :449-58 - DOI:10.1007/s00216-010-4346-8 - 2011
Two molecularly imprinted silicas (MISs) were synthesized and used as selective sorbents for the extraction of nitroaromatic explosives in post-blast samples. The synthesis of the MISs was carried out with phenyltrimethoxysilane as monomer, 2,4-dinitrotoluene (2,4-DNT) as template and triethoxysilane as cross-linker by a sol-gel approach in two molar ratios: 1/4/20 and 1/4/30 (template/monomer/cross-linker). Non-imprinted silica sorbents were also prepared following the same procedures without introducing the template. An optimized procedure dedicated to the selective treatment of aqueous samples was developed for both MISs for the simultaneous extraction of the template and other nitroaromatic compounds commonly used as explosives. The capacity of the MISs was measured by the extraction of increasing amounts of 2,4-DNT in pure water and is higher than 3.2 mg/g of sorbent for each MIS. For the first time, four nitroaromatic compounds were selectively extracted and determined simultaneously with extraction recoveries higher than 79%. The potential of these sorbents was then highlighted by their use for the clean-up of post-blast samples (motor oil, post-mortem blood, calcined fragments, etc.). The results were compared to those obtained using a conventional sorbent, thus demonstrating the interest of the use of these MISs as selective sorbents.
Surface Reactivity from Electrochemical Lithography: Illustration in the Steady-State Reductive Etching of Perfluorinated Surfaces
H. Hazimeh, S. Nunige, R. Cornut, C. Lefrou, C. Combellas, F. Kanoufi
Anal. Chem. - 83(15) :6106-13 - DOI:10.1021/ac201255c - 2011
The scanning electrochemical microscope (SECM) in the lithographic mode is used to assess quantitatively, from both theoretical and experimental points of view, the kinetics of irreversible transformation of electroactive molecular moieties immobilized on a surface as self-assembled monolayers (SAMs). The SECM tip allows the generation of an etchant that transforms the surface locally and irreversibly. The resulting surface patterning is detectable by different surface analyses. The quantification of the surface transformation kinetics is deduced from the evolution of the pattern dimensions with the etching time. The special case of slow etching kinetics is presented; it is predicted that the pattern evolution follows the expansion of the etchant at the substrate surface. The case of a chemically unstable etchant is considered. The model is then tested by inspecting the slow reductive patterning of a perfluorinated SAM. Good agreement is found with different independent SECM interrogation modes, depending on the insulating or conducting nature of the covered substrate. The surface transformation measurements are also compared to the reduction of solutions of perfluoroalkanes. The three-orders-of-magnitude-slower electron transfer observed at the immobilized molecules likely describes the large reorganization associated with the generation of a perfluoroalkyl-centered radical anion.
Feasibility of ultra high performance supercritical neat carbon dioxide. chromatography at conventional pressures
C. Sarazin, D. Thiebaut, P. Sassiat,J. Vial
J. Sep. Sci. - 34(19) :2773-8 - DOI:10.1002/jssc.201100332 - 2011
The implementation of columns packed with sub-2 µm particles in supercritical fluid chromatography (SFC) is described using neat carbon dioxide as the mobile phase. A conventional supercritical fluid chromatograph was slightly modified to reduce extra column band broadening. Performances of a column packed with 1.8 µm C18-bonded silica particles in SFC using neat carbon dioxide as the mobile phase were compared with results obtained in ultra high performance liquid chromatography (UHPLC) using a dedicated chromatograph. As expected and usual in SFC, higher linear velocities than in UHPLC must be applied in order to reach optimal efficiency owing to higher diffusion coefficient of solutes in the mobile phase; similar numbers of theoretical plates were obtained with both techniques. Very fast separations of hydrocarbons are presented using two different alkyl-bonded silica columns.
Integrated and Diffusion-Based Micro-Injectors for Open Access Cell Assays
X. Li, L. Liu, L. Wang, K. Kamei, Q. H. Yuan, F. Zhang, J. Shi, A. Kusumi, M. Xie, Z. J. Zhao and Y. Chen
Lab. Chip - 11 :2612-7 - DOI:10.1039/c1lc20258h - 2011
Currently, most microfluidic devices are fabricated with embedded micro-channels and other elements in a close form with outward connections. Although much functionality has been demonstrated and a large number of applications have been developed, they are not easy for routine operation in biology laboratories where most in vitro cell processing still relies on the use of culture dishes, glass slides, multi-well plates, tubes, pipettes, etc. We report here an open access device which consists of an array of isolated micro-channels plated on a large culture surface, each of them having tiny nozzles for localized drug delivery. In a diffusion dominant regime, steady gradients of molecule concentration could be obtained and varied by changing the flow rate inside the micro-channels. As assay examples, cell staining and drug-induced cell apoptosis were demonstrated, showing fast cell responses in close proximity of the nozzles.
Photoreversible Fragmentation of a Liquid Interface for Micro-Droplet Generation by Light Actuation
A. Diguet, H. Li, N. Queyriaux, Y. Chen and D. Baigl
Lab. Chip - 11 :2666-9 - DOI:10.1039/c1lc20328b - 2011
We describe a method to induce by light a reversible switch from a continuous two-phase laminar flow to a droplet generating regime, in microfluidic devices with a usual water-in-oil flow focusing geometry. It consists in adding a photosensitive surfactant to the aqueous phase to modulate using light the interfacial energy between flowing liquids and the microfluidic substrate. We show that UV irradiation induces liquid fragmentation into monodisperse water microdroplets and that many cycles of reversible and rapid switches (<2 s) between continuous laminar flows and stable droplet regimes can be realized. By spatially controlling the application of the light stimulus, we also demonstrate the first spatially resolved remote induction of droplet generation.


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