Combination of COFRADIC and high temperature-extended column length conventional liquid chromatography: a very efficient way to tackle complex protein samples, such as serum

The previously reported COmbined FRActional DIagonal Chromatography (COFRA-DIC) methodology, in which a subset of peptides representative for their parent proteins are sorted, is particularly powerful for whole proteome analysis. This peptide-centric technology is built around diagonal chromatography, where peptide separations are crucial. This paper presents high efficiency peptide separations, in which four 250 x 2.1 mm, 5 microm Zorbax 300SB-C18 columns (total length 1 m) were coupled at operating temperatures of 60'C using a dedicated LC oven and conventional LC equipment. The high efficiency separations were combined with the COFRADIC procedure. This extremely powerful combination resulted, for the analysis of serum, in an increase in the uniquely identified peptide sequences by a factor of 2.6, compared to the COFRADIC procedure on a 25 cm column. This is a reflection of the increased peak capacity obtained on the 1 m column, which was calculated to be a factor 2.7 higher than on the 25 cm column. Besides more efficient sorting, less ion suppression was noticed.     

Assessing a novel microfluidic interface for shotgun proteome analyses

Microfluidic interfaces coupled to ESI mass spectrometers hold great potential for proteomics as they have been shown to augment the overall sensitivity of measurements and require only a minimum of operator manipulations as compared to conventional nano-LC interfaces. Here, we evaluated a new type of HPLC-Chips holding larger enrichment columns (thus an increased sample loading capacity) for gel-free proteome studies. A tryptic digest of a human T-cell proteome was fractionated by strong cation exchange chromatography and selected fractions were analyzed by MS/MS on an IT mass spectrometer using both the new HPLC-Chip as well as a conventional nano-LC-MS/MS interface. Our results indicate that the HPLC-Chip is capable of handling very complex peptide mixtures and, in fact, leads to the identification of more peptides and proteins as compared to when a conventional interface was used. The HPLC-Chip preferentially produced doubly charged tryptic peptides. We further show that MS/MS spectra of doubly charged tryptic peptide ions are more readily identified by MASCOT as compared to those from triply charged precursors and thus argue that besides the improved chromatographic conditions provided by the HPLC-Chip, its peptide charging profile might be a secondary factor leading to an increased proteome coverage.     

A critical comparison of analytical flow systems exploiting streamlined and pulsed flows

Multipumping (MPFS) and multicommuted (MCFS) flow systems relying on pulsed and laminar flows were critically compared. The mixing conditions and dispersion associated with both systems were evaluated by simulating the sample with bromocresol green. The molybdenum blue method for phosphate determination in soil extracts was also implemented in both flow systems. Furthermore, laser-induced fluorescence (LIF) was applied to visualize the dispersing sample; rhodamine B was used as the fluorescent species. The pulsed flow enhanced the mixing of the solutions involved, thus reducing reagent consumption (48 and 96 microl for MPFS and MCFS), and improving sampling rate (67 and 144 h(-1) for MCFS and MPFS). For phosphate determination, results obtained with both systems were precise (r.s.d. < 0.5%; n = 10) and accurate. Analyses of the absorbance vs time/space LIF plots revealed that exploitation of pulsed flow led to a pronounced radial dispersion and to a limited axial dispersion, typical aspects of turbulent flows.     

Structure of sodium carboxymethyl cellulose aqueous solutions: A SANS and rheology study

We report a small angle neutron scattering (SANS) and rheology study of cellulose derivative polyelectrolyte sodium carboxymethyl cellulose with a degree of substitution of 1.2. Using SANS, we establish that this polymer is molecularly dissolved in water with a locally stiff conformation with a stretching parameter . We determine the cross sectional radius of the chain ( 3.4 Å) and the scaling of the correlation length with concentration (ξ = 296 c^?1∕2Å for c in g/L) is found to remain unchanged from the semidilute to concentrated crossover as identified by rheology. Viscosity measurements are found to be in qualitative agreement with scaling theory predictions for flexible polyelectrolytes exhibiting semidilute unentangled and entangled regimes, followed by what appears to be a crossover to neutral polymer concentration dependence of viscosity at high concentrations. Yet those higher concentrations, in the concentrated regime defined by rheology, still exhibit a peak in the scattering function that indicates a correlation length that continues to scale as . © 2014 The Authors. Journal of Polymer Science Part B: Polymer Physics Published by Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2015 , 53, 492–501     

An N‐Acetyl Cysteine Ruthenium Tricarbonyl Conjugate Enables Simultaneous Release of CO and Ablation of Reactive Oxygen Species

We have designed and synthesised a [Ru(CO)₃Cl₂(NAC)] pro‐drug that features an N‐acetyl cysteine (NAC) ligand. This NAC carbon monoxide releasing molecule (CORM) conjugate is able to simultaneously release biologically active CO and to ablate the concurrent formation of reactive oxygen species (ROS). Complexes of the general formulae [Ru(CO)₃(L)₃]²⁺, including [Ru(CO)₃Cl(glycinate)] (CORM‐3), have been shown to produce ROS through a water–gas shift reaction, which contributes significantly, for example, to their antibacterial activity. In contrast, NAC‐CORM conjugates do not produce ROS or possess antibacterial activity. In addition, we demonstrate the synergistic effect of CO and NAC both for the inhibition of nitric oxide (formation) and in the expression of tumour‐necrosis factor (TNF)‐α. This work highlights the advantages of combining a CO‐releasing scaffold with the anti‐oxidant and anti‐inflammatory drug NAC in a unique pro‐drug.     

Coating on Zinc Surface to Improve the Electrochemical Behavior of Zinc Anodes for Zinc‐Air Fuel Cells

The corrosion of Zn anode, associated with corrosion current and hydrogen evolution reaction, is a major reason for capacity loss and safety problems. As a way of improving the current issue, a newly coating method using difference of melting points has been attempted to synthesize anode materials and found to be highly effective. Effects of corrosion inhibitors are also investigated by diverse electrochemical techniques. The Tafel extrapolation shows the reduced corrosion current compared with that of pure Zn. ZnNi‐2 (Zn:Ni, 95 : 5 wt.%) demonstrates the hydrogen overpotential is shifted to the most negative value as ?1.7 V vs. Hg/HgO.     

Analysis of the cyanolichen Lichina pygmaea metabolites using in situ DART‐MS: from detection to thermochemistry of mycosporine serinol

Direct Analysis in Real Time DART‐HRMS is here first applied to the detection of molecules from a lichen, Lichina pygmaea. The aim was to propose an innovative method of in situ detection of lichen secondary metabolites using the possibilities of elemental composition determination available when a DART source is interfaced with a TOF analyzer. Three kinds of samples have been submitted to DART ionization, i.e. an intact thallus, a powder obtained from the crushed lichen and an aqueous extract. In situ analysis of crushed lichen, yields an extensive chemical profile, comparable to what is obtained from the aqueous extract, comprising both major polar metabolites described in literature along with some other signals that could correspond to potentially unknown metabolites. One of the detected secondary metabolites, mycosporine serinol, underwent a dehydration reaction prior to its transfer in the gas‐phase by DART ionization. The consideration of the thermal transfers involved in the DART ionization process and the possibility to record time‐dependent mass spectra through the use of the TOF analyzer allowed establishing Arrhenius plots of this water molecule loss to obtain associated thermodynamic quantities. The low values of corresponding activation enthalpy ( of the order of 25 kJ mol^?1) enabled formulating some assumption regarding a possible role of such metabolites in the lichen. Copyright © 2015 John Wiley & Sons, Ltd.     

Size‐Controlled Construction of Magnetic Nanoparticle Clusters Using DNA‐Binding Zinc Finger Protein

Nanoparticle clusters (NPCs) have attracted significant interest owing to their unique characteristics arising from their collective individual properties. Nonetheless, the construction of NPCs in a structurally well‐defined and size‐controllable manner remains a challenge. Here we demonstrate a strategy to construct size‐controlled NPCs using the DNA‐binding zinc finger (ZnF) protein. Biotinylated ZnF was conjugated to DNA templates with different lengths, followed by incubation with neutravidin‐conjugated nanoparticles. The sequence specificity of ZnF and programmable DNA templates enabled a size‐controlled construction of NPCs, resulting in a homogeneous size distribution. We demonstrated the utility of magnetic NPCs by showing a three‐fold increase in the spin–spin relaxivity in MRI compared with Feridex. Furthermore, folate‐conjugated magnetic NPCs exhibited a specific targeting ability for HeLa cells. The present approach can be applicable to other nanoparticles, finding wide applications in many areas such as disease diagnosis, imaging, and delivery of drugs and genes.     

Controlling Internal Pore Sizes in Bicontinuous Polymeric Nanospheres

Complex polymeric nanospheres were formed in water from comb‐like amphiphilic block copolymers. Their internal morphology was determined by three‐dimensional cryo‐electron tomographic analysis. Varying the polymer molecular weight (MW) and the hydrophilic block weight content allowed for fine control over the internal structure. Construction of a partial phase diagram allowed us to determine the criteria for the formation of bicontinuous polymer nanosphere (BPN), namely for copolymers with MW of up to 17 kDa and hydrophilic weight fractions of ≤0.25; and varying the organic solvent to water ratio used in their preparation allowed for control over nanosphere diameters from 70 to 460 nm. Significantly, altering the block copolymer hydrophilic–hydrophobic balance enabled control of the internal pore diameter of the BPNs from 10 to 19 nm.     

Controlling the Formation of Ionic‐Liquid‐based Aqueous Biphasic Systems by Changing the Hydrogen‐Bonding Ability of Polyethylene Glycol End Groups

The formation of aqueous biphasic systems (ABS) when mixing aqueous solutions of polyethylene glycol (PEG) and an ionic liquid (IL) can be controlled by modifying the hydrogen‐bond‐donating/‐accepting ability of the polymer end groups. It is shown that the miscibility/immiscibility in these systems stems from both the solvation of the ether groups in the oxygen chain and the ability of the PEG terminal groups to preferably hydrogen bond with water or the anion of the salt. The removal of even one hydrogen bond in PEG can noticeably affect the phase behavior, especially in the region of the phase diagram in which all the ethylene oxide (EO) units of the polymeric chain are completely solvated. In this region, removing or weakening the hydrogen‐bond‐donating ability of PEG results in greater immiscibility, and thus, in a higher ability to form ABS, as a result of the much weaker interactions between the IL anion and the PEG end groups.