Capillary Electrophoresis as a Second Dimension to Isoelectric Focusing for Peptide Separation

Capillary zone electrophoresis and carrier ampholytes based capillary electrophoresis have been used as a second separation step to Off-Gel isoelectric focusing for the analysis of complex peptide mixtures. A tryptic digest of four proteins (bovine serum albumin, β-lactoglobulin, horse myoglobin, cytochrome c) has been chosen as a peptide test mixture. After assessment of different modes of capillary electrophoresis as a second dimension to Off-Gel isoelectric focusing, the optimized two-dimensional platforms provide a degree of orthogonality comparable to state-of-the-art multidimensional liquid chromatography systems as well as a practical peak capacity above 700

Electrokinetic supercharging for highly efficient peptide preconcentration in capillary zone electrophoresis

Electrokinetic supercharging has been integrated in CZE for the development of a highly sensitive methodology for protein tryptic digest analysis. A careful choice of the experimental conditions led to sensitivity enhancement factors between 1000 and 10 000 whilst maintaining a satisfactory resolution. Peptides in the low nanomolar concentration range have been detected despite the use of the poorly sensitive UV absorbance detection mode. The buffer system used in this study is fully suitable for coupling CE to MS

Iontophoretic Fraction Collection for Coupling Capillary Zone Electrophoresis with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

An automated fraction collection interface has been developed for coupling capillary electrophoresis (CE) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). This fraction collection approach is based on electromigration and diffusion and does not rely on the presence of a liquid junction, sheath-liquid, electro-osmotic flow, or a superimposed hydrodynamic flow. Neutrally coated capillary with negligible electroosmosis can thus be used to provide high-efficiency separations of biological compounds. The in-capillary separation resolution is totally independent from the spotting process. CE-separated species can be collected either in a multiwell plate or directly on a MALDI target. In the present work, an eight-protein mixture, submitted to trypsin proteolysis, has been used as a sample test and separations have been conducted in 50 μm i.d. neutrally coated capillaries. As compared to direct MALDI MS analysis, the integration of CE improved the number of detected peptides from 36 to 87 and the average sequence coverage from 24% to 38%. Internal calibration was used, and an average mass accuracy of 16.1 ppm is reported. Finally, diffusion-migration numerical simulations of the iontophoretic fraction collection process have been carried out

Adsorbed protein detection by scanning electrochemical microscopy

A scanning electrochemical microscopy (SECM) protein detection methodology has been developed based on the tagging of free cysteines and other nucleophiles in proteins and peptides by benzoquinone. The tagged proteins are detected by the mediated reduction of benzoquinone with a redox species produced electrochemically at the SECM tip. After careful optimization, a sensitivity in the low ng mm 2 range was reached for bovine serum albumin. One of the major advantages of the present technique is that the selectivity of the protein tagging can be tuned by changing the pH of the reaction media. Depending on the requirements, cysteine selective or general detection can therefore be achieved with a high sensitivity. As a proof of concept, this technique was applied to the detection of protein spots and to the imaging of human fingerprints and further compared to the actual SECM state-of-art approach

In-source photocatalytic reduction of disulfide bonds during laser desorption ionization

A photosensitive plate based on sintered TiO2 nanoparticles has been developed to carry out in-source photo-induced reductions for cleavage of disulfide bridges using glucose as a hole scavenger during laser desorption ionization

Magnetic Beads Based Immunoaffinity Capillary Electrophoresis of Total Serum IgE with Laser-Induced Fluorescence Detection

A magnetic beads based immunoaffinity capillary electrophoresis method for total Immunoglobulin E quantification in serum has been developed. The method combines speed, automation ability, and minimal sample consumption. Only 1 μL of serum is required while the whole immunoaffinity capillary electrophoresis method is performed in less than 50 min. The concomitant use of online immunocapture, transient isotachophoresis, and laserinduced fluorescence detection provides a sensitivity in the low picomolar range and a highly linear fluorescence response over 4 orders of magnitude (IgE concentration ranging from 2.4 to 2400 ng/mL). After validation with a reference material, the method has been successfully applied to the quantification of total IgEs in patient sera. The results compared well with classical ImmunoCap data