DMF-MALDI: droplet based microfluidic combined to MALDI-TOF for focused peptide detection.

We present an automated droplet microfluidic system (DMF) to generate monitored nanoliter aqueous droplets in oil and their deposition on a commercial stainless steel plate for MALDI-TOF analysis of peptides or protein digests. We demonstrate that DMF-MALDI combination focuses the analyte on the MALDI plate, increasing considerably the homogeneity of the dried material. This results in a 30times enhanced MALDI-TOF MS signal for a model peptide, allowing a significant improvement of the detection sensitivity limit (down to few tens of attomoles). Moreover, positive detection can be achieved from sub-nanomolar peptides solutions and better overall protein sequence coverages are obtained from few tens attomoles of protein digest. These results make DMF-MALDI a promising approach for the treatment of peptides samples as well as a key component for an integrated approach in the proteomic field.

Capillary electrophoretic focusing of covalently derivatized protein induced by surfactant.

In this communication, we present a very simple strategy to focus covalently derivatized proteins for high sensitivity CE analysis by LIF detection. We demonstrated that the covalently tagged protein can be focused just by adding SDS at a concentration above the CMC in the derivatized sample. Under specific injection conditions, SDS concentration below the CMC is also sufficient to induce the focusing of the tagged protein. This method allows the quantification and detection of the covalently tagged protein in a narrow zone with an efficiency approaching 220 000 plates/m. Very good linearity was obtained for the ubiquitin in a concentration range of 2-25 µM.

Neutral polymers as coatings for high resolution electrophoretic separation of Aß peptides on glass microchips.

This study reports a comparison of the performances of two neutral polymers, poly ethylene-oxide (PEO) and poly(dimethylacrylamide-co-allyl glycidyl ether) (EpDMA), in glass microchips to achieve zone electrophoresis separation of several truncated forms of beta amyloid (Aß) peptides, sharing very similar structures. The peptides were derivatized by FluoProbes 488 NHS to allow their fluorescence detection. Two protocols based either on PEO or EpDMA led to good pH stabilities in addition to a significant reduction of the electroosmotic flow. These two polymer coatings allowed repeatable analyses and high resolution for the simultaneous analysis of three Aß peptides, Aß 1-38, Aß 1-40 and Aß 1-42, considered as potential biomarkers of Alzheimer's disease. A recovery study showed that EpDMA was superior in reducing the adsorption of the Aß peptides on the coated inner wall. Finally, the separation method relying on the EpDMA coated microchips was validated as linear using a calibration curve and the LOD was estimated to be close to 200 nM. Despite very short migration distances, different N-terminal or C-terminal truncated Aß peptides, corresponding to promising biomarker combinations for the future diagnostic, were fully resolved. The method was successfully applied to detect these peptides in spiked cerebrospinal fluid and has provided a first achievement towards the development of a microsystem that would integrate preconcentration and separation steps.

On-line capillary electrophoresis derivatization method for high sensitivity analysis of ubiquitin in filtered cerebrospinal fluid.

Electrophoretically mediated microanalysis was implemented for on-line fluorescence derivatization of ubiquitin (UBI), a potential biomarker of Alzheimer disease. Ubiquitin was labeled on its amino groups by the Fluoprobes® 488 N-hydroxysuccinimide. The pH of the BGE, the applied electric field, and the washing process of the capillary were optimized. The best derivatization yield was obtained at pH 9.5 under an electric field of ∼210 V/cm. The concentration and volume of the fluorescent dye as well as the sample medium and volume of injected UBI were also optimized. In order to improve further the LOD of UBI, electrophoretically mediated microanalysis was performed in a wider (100 µm id) and longer (110 cm) capillary. As expected, these capillary dimensions improved the analytical sensitivity of UBI when compared to the 50 µm id capillary. Under the optimized conditions, this analytical methodology allowed the analysis of free UBI in standard solution with a LOD of ∼15 nM. Finally, the on-line fluorescent derivatization of UBI was applied to the detection and the quantification of UBI in cerebrospinal fluid (CSF) samples. Due to the high salt concentration of biological samples, desalting of CSF was required prior to the CE analysis. Quantification of UBI in CSF either by a direct evaluation of peak areas or using the standard addition method was achieved.

Contribution of CE to the analysis of protein or peptide biomarkers.

Biomarker analysis is pivotal for disease diagnosis and one important class of biomarkers is constituted by proteins and peptides. This review focuses on protein and peptide analyses from biological fluids performed by capillary electrophoresis. The various strategies that have been reported to prevent difficulties due to the handling of real samples are described. Innovative techniques to overcome the complexity of the sample, to prevent the adsorption of the analytes on the inner capillary wall, and to increase the sensibility of the analysis are summarized and illustrated by different applications. To fully illustrate the contribution of CE to the analysis of biomarkers from human sample, two detailed protocols are given: the analysis from CSF of five amyloid peptide, biomarkers of the Alzheimer disease, and the analysis of sialoforms of transferrin from human serum.