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We present a comprehensive method for proteome analysis that integrates both intact protein separation and proteolytic fragment characterization mass spectrometric approaches. Strong cation exchange chromatography (SCX) was used as the first separation dimension and capillary reversed-phase liquid chromatography (cRPLC) was integrated as the second separation dimension. Fractions from SCX were collected offline and loaded onto cRPLC. Effluents from cRPLC were directly deposited onto the MALDI target plates and further digested by using a rapid on-probe tryptic digestion technique. This approach minimizes the amount of time and extensive labor required for traditional in-solution digestion followed by exhaustive sample cleanup and transfer. MALDI-TOF/TOF was used for subsequent analyses. The sensitivity of on-target digestion is showed by analyzing 0.07 ng of myoglobin, 0.07 ng of cytochrome c and 0.7 ng BSA. The high efficiency of the overall system was demonstrated by the analysis of intact proteins extracted from normal human liver tissue. In total, 458 proteins were identified, which proved the system's promising potential for analysis and application in proteomics. [ABSTRACT FROM AUTHOR]

The cationic surfactants didodecyldimethylammonium bromide (DDAB) and dioctadecyldimethyl-ammonium bromide (DODAB) have previously been shown to form semi-permanent coatings that effectively prevent adsorption of cationic proteins in fused silica capillaries with inner diameters of 25–75 µm. This paper investigates the impact that narrower capillary diameters (≤25 µm) have on the stability of surfactant bilayer coatings and the efficiency of separations of model cationic proteins and neurotransmitters. Using a DODAB-coated 5 µm i.d. capillary 210 consecutive protein separations (1050 min) were performed without recoating the capillary between runs. Separation efficiencies of 1 400 000–2 000 000 plates per m (340 000–430 000 plates) were obtained. Migration time reproducibilites of 6.8% RSD were observed for 300 injections performed over a 30 day period without any regeneration of the coating. Neurotransmitters were separated with efficiencies ranging from 470 000-–610 000 plates per m (110 000–140 000 plates) in a 5 µm capillary. [ABSTRACT FROM AUTHOR]

This paper describes the detection of a cardiac biomarker, cardiac troponin I (cTnI), spiked into depleted human serum using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic channel. The microfluidic chip incorporates a 100× cross-sectional area reduction, including a 10× depth reduction and a 10× width reduction, to increase sensitivity during ITP. The cross-sectional area reductions in combination with ITP allowed visualization of lower concentrations of fluorescently labeled cTnI. ITP was performed in both “peak mode” and “plateau mode” and the final concentrations obtained were linear with initial cTnI concentration. We were able to detect and quantify cTnI at initial concentrations as low as 46 ng mL−1in the presence of human serum proteins and obtain cTnI concentrations factors as high as ∼ 9000. In addition, preliminary ITP experiments including both labeled cTnI and labeled protein kinase A (PKA) phosphorylated cTnI were performed to visualize ITP migration of different phosphorylated forms of cTnI. The different phosphorylated states of cTnI formed distinct ITP zones between the leading and terminating electrolytes. To our knowledge, this is the first attempt at using ITP in a cascade microchip to quantify cTnI in human serum and detect different phosphorylated forms. [ABSTRACT FROM AUTHOR]

Due to their unique physical, chemical and optical properties, quantum dots (QDs) have been widely applied in biological and biomedical research. However, the influence of cations on QDs has rarely been explored except for the quenching effect on QD fluorescence by some heavy metal ions (e.g.Ag+ions and Cu2+ions). Here we present a new approach to study the effect of physiological cations (e.g.Ca2+ions, Mg2+ions, Na+ions and K+ions) upon QDs by using single-particle detection. Our results demonstrate that divalent cations (e.g.Ca2+ions and Mg2+ions) can selectively induce the self-assembly of QDs in a concentration- and time-dependent manner, which is impossible to attain with ensemble fluorescence measurements. In comparison with conventional TEM, SEM and AFM measurements, this single-particle detection has significant advantages of simple/no sample preparation, rapid analysis, low cost, and easy measurement in the native environment. This single-particle detection technique might also provide a unique approach to study enzyme-responsive bimolecular assembly, and the characterization of early protein assembly states. [ABSTRACT FROM AUTHOR]

A reconstituted myoglobin with a synthetic cofactor having anionic binding sites effectively works as a photocatalyst for hydrogen generation in the presence of monomethylated bipyridinium. [ABSTRACT FROM AUTHOR]