Programme Hospitalier Recherche Clinique, Institut Pasteur, Inserm, French Public Health Agency.

We present the state-of-the-art in miniaturized sample preparation, immunoassays, one-dimensional and multidimensional analyte separations, and coupling of microdevices with electrospray ionization-mass spectrometry. Hyphenation of these different techniques and their relevance to proteomics will be discussed. In particular, we will show that analytical performances of microfluidic analytical systems are already close to fulfill the requirements for proteomics, and that miniaturization results at the same time in a dramatic increase in analysis throughput. Throughout this review, some examples of analytical operations that cannot be achieved without microfluidics will be emphasized. Finally, conditions for the spreading of microanalytical systems in routine proteomic labs will be discussed.

Hyperpolarized substrates prepared via dissolution dynamic nuclear polarization have been proposed as magnetic resonance imaging (MRI) agents for cancer or cardiac failure diagnosis and therapy monitoring through the detection of metabolic impairments in vivo. The use of potentially toxic persistent radicals to hyperpolarize substrates was hitherto required. We demonstrate that by shining UV light for an hour on a frozen pure endogenous substance, namely the glucose metabolic product pyruvic acid, it is possible to generate a concentration of photo-induced radicals that is large enough to highly enhance the 13 C polarization of the substance via dynamic nuclear polarization. These radicals recombine upon dissolution and a solution composed of purely endogenous products is obtained for performing in vivo metabolic hyperpolarized 13 C MRI with high spatial resolution. Our method opens the way to safe and straightforward preclinical and clinical applications of hyperpolarized MRI because the filtering procedure mandatory for clinical applications and the associated pharmacological tests necessary to prevent contamination are eliminated, concurrently allowing a decrease in the delay between preparation and injection of the imaging agents for improved in vivo sensitivity.is a very powerful imaging modality in terms of temporal and spatial resolution of anatomical structures. The modality is widespread, well established in clinical environments, and paramagnetic agents are used extensively for enhanced contrast or perfusion examination. MR is also a unique technique to obtain in vivo metabolic maps using the spectroscopic information that can be extracted from the time-domain acquisitions. In particular, it is possible to monitor the biochemical transformations of specific substrates that are delivered to subjects. Because it gives access to the kinetics of the conversion of substrates into metabolites, MR spectroscopy (MRS) of the carbon nuclei ( 13 C) is one of the most powerful techniques to investigate intermediary metabolism (1).The well-known weakness of MR as a spectroscopic technique is its relatively low sensitivity. It can be offset by so-called hyperpolarization methods, in particular the one based on dynamic nuclear polarization (DNP) (2), which is now commonly referred to as dissolution DNP and was first proposed about a decade ago (3). The hyperpolarized substrates obtained following dissolution DNP are biomolecules in aqueous solution with a largely out-ofequilibrium nuclear spin polarization corresponding to an enhancement of several orders of magnitude compared with the thermal equilibrium polarization attainable in MRI scanners. A basic requirement for DNP is the presence of unpaired electron spins in the sample to be hyperpolarized. These polarizing agents are usually incorporated in the form of persistent radicals. An inherent limit of any hyperpolarization method is that the intrinsic longitudinal nuclear spin relaxation will annihilate the polarization enhancement in the course of time to reach t...

An on-plate specific enrichment method is presented for the direct analysis of peptides phosphorylation. An array of sintered TiO 2 nanoparticle spots was prepared on a stainless steel plate to provide porous substrate with a very large specific surface and durable functions. These spots were used to selectively capture phosphorylated peptides from peptide mixtures, and the immobilized phosphopeptides could then be analyzed directly by MALDI MS after washing away the nonphosphorylated peptides. beta-Casein and protein mixtures were employed as model samples to investigate the selection efficiency. In this strategy, the steps of phosphopeptide capture, purification, and subsequent mass spectrometry analysis are all successfully accomplished on a single target plate, which greatly reduces sample loss and simplifies analytical procedures. The low detection limit, small sample size, and rapid selective entrapment show that this on-plate strategy is promising for online enrichment of phosphopeptides, which is essential for the analysis of minute amount of samples in high-throughput proteome research.

Hyperpolarization via dynamic nuclear polarization (DNP) is pivotal for boosting magnetic resonance imaging (MRI) sensitivity and dissolution DNP can be used to perform in vivo real-time 13C MRI. The type of applications is however limited by the relatively fast decay time of the hyperpolarized spin state together with the constraint of having to polarize the 13C spins in a dedicated apparatus nearby but separated from the MRI magnet. We herein demonstrate that by polarizing 13C with photo-induced radicals, which can be subsequently annihilated using a thermalization process that maintains the sample temperature below its melting point, hyperpolarized 13C-substrates can be extracted from the DNP apparatus in the solid form, while maintaining the enhanced 13C polarization. The melting procedure necessary to transform the frozen solid into an injectable solution containing the hyperpolarized 13C-substrates can therefore be performed ex situ, up to several hours after extraction and storage of the polarized solid.

TiO 2 /ITO modified electrodes were developed to quantitatively photo-oxidise adsorbed ds-DNA and to study the effect of antioxidants as ds-DNA protecting agents. TiO 2 films are used for efficient ds-DNA immobilization, for ds-DNA oxidation through photogenerated hydroxyl radicals and as electrode for amperometric sensing. The films, prepared by a sol-gel process, are deposited on ITO glass electrodes. Damages occurring after ds-DNA oxidation by ROS, are detected by adding MB as an intercalant probe and by monitoring the electrochemical reduction current of the intercalated redox probe. The MB electrochemical signal is found to be sensitive enough to monitor ds-DNA structure changes and the electrochemical sensor has been applied to the evaluation of the antioxidant properties of glutathione and gallic acid.

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Hyperpolarization via dissolution dynamic nuclear polarization (DNP) is a versatile method to dramatically enhance the liquid-state NMR signal of X-nuclei and can be used for performing metabolic and molecular imaging. It was recently demonstrated that instead of incorporating persistent radicals as source of unpaired electron spins, required for DNP, nonpersistent radicals can be photoinduced in frozen beads of neat pyruvic acid (PA), the most common substrate for metabolic imaging. In the present work, it is shown that the same radicals can be created in frozen solutions containing a fraction of PA in addition to 13 C-or 6 Li-labeled salts or 129 Xe nuclei. The use of these nonpersistent radicals prevents the loss of a substantial part of the polarization during the transfer of hyperpolarized solutions into iron-shielded high-field MRI scanners. It is also demonstrated that UV-irradiated d 4 -PA yields nonpersistent radicals exhibiting similarities with the most efficient and widely used persistent trityl radicals.