Capillary liquid chromatography interfaced to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using an on-line coupled piezoelectric flow-through microdispenser

A piezoelectric flow-through microdispenser interfacing capillary liquid chromatography (LC) with matrix-assisted laser desorption/ionization time-of-fight mass spectrometry (MALDI-TOF MS) was developed for the identification of biomolecules. The MALDI target plate was placed on a computer controlled high-resolution x-y stage, on to which the column effluent was deposited as discrete spots, which thereby facilitated tracing of the chromatographic separation. The entire target plate was sprayed with a homogeneous layer of alpha-cyano-4-cinnamic acid mixed with nitrocellulose by using an air-brush. Hence the tedious manual handling of a micropipetter applying matrix solution on top of each fraction collected spot was avoided. The pre-made target plates were stable for at least 3 weeks if kept in darkness at room temperature, which easily allowed re-analysis of dispensed sample spots. The integrated microsystem was characterized and optimized by means of fluidics, dispersion, operational stability and sensitivity parameters. The dispensing unit was developed specifically to match high-resolution capillary LC separations using a dispenser with an internal volume from inlet to the ejecting nozzle of 250 nl. Minimizing dead volumes was crucial in order to maintain the chromatographic resolution. The volume of the ejected droplets was of the order of 60 pl. Successful separations of seven immunoregulating peptides were made: ACTH 1-17, bradykinin, enkephalin, angiotensin III, angiotensin II, angiotensin I and ACTH 18-39. On-line sample dispensing on the target plate in combination with trace enrichment followed by automated MALDI-TOF MS identification is demonstrated, reaching a sensitivity of 100 amol.     

Coulometric titration of deactivated phenols with bromine

A number of deactivated phenols containing fluorine, chlorine or bromine, formyi, acetyl, carboxyl or nitro groups have been titrated with anodically generated bromine. The reaction was carried out in a water-acetic acid-pyridine medium and the reactivity was controlled by varying the water and pyridine content and the concentration of bromide ion. Hydrogen in all free positions ortho and para to the phenolic hydroxyl group is generally exchanged for bromine, but in certain instances a partial bromination is possible. The method as developed is widely applicable for deactivated phenols. Only certain ortho-substituted phenols could not be quantitatively titrated. The mean relative error for the phenols titrated was ± 1·2%.     

Dynamics in atomistic simulations of phospholipid membranes: Nuclear magnetic resonance relaxation rates and lateral diffusion

It is shown that a long, near microsecond, atomistic simulation can shed some light upon the dynamical processes occurring in a lipid bilayer. The analysis focuses on reorientational dynamics of the chains and lateral diffusion of lipids. It is shown that the reorientational correlation functions exhibits an algebraic decay (rather than exponential) for several orders of magnitude in time. The calculated nuclear magnetic resonance relaxation rates agree with experiments for carbons at the C7 position while there are some differences for C3. Lateral diffusion can be divided into two stages. In a first stage occurring at short times, t<5 ns, the center of mass of the lipid moves due to conformational changes of the chains while the headgroup position remains relatively fixed. In this stage, the center of mass can move up to approximately 0.8 nm. The fitted short-time diffusion coefficient is D(1)=13 x 10(-7) cm(2) s(-1) On a longer time scale, the diffusion coefficient becomes D(2)=0.79 x 10(-7) cm(2) s(-1).     

Vertex and edge spread of zero forcing number,maximum nullity,and minimum rank of a graph

The minimum rank of a simple graph G is defined to be the smallest possible rank over all symmetric real matrices whose ijth entry (for $i\ne j$) is nonzero whenever $\{i\text{,}j\}$ is an edge in G and is zero otherwise; maximum nullity is taken over the same set of matrices. The zero forcing number is the minimum size of a zero forcing set of vertices and bounds the maximum nullity from above. The spread of a graph parameter at a vertex v or edge e of G is the difference between the value of the parameter on G and on $G-v$ or $G-e$. Rank spread (at a vertex) was introduced in [4]. This paper introduces vertex spread of the zero forcing number and edge spreads for minimum rank/maximum nullity and zero forcing number. Properties of the spreads are established and used to determine values of the minimum rank/maximum nullity and zero forcing number for various types of grids with a vertex or edge deleted.     

Effect of different treatments of long-range interactions and sampling conditions in molecular dynamic simulations of rhodopsin embedded in a dipalmitoyl phosphatidylcholine bilayer

The present study analyzes the effect of the simulation conditions on the results of molecular dynamics simulations of G-protein coupled receptors (GPCRs) performed with an explicit lipid bilayer. Accordingly, the present work reports the analysis of different simulations of bovine rhodopsin embedded in a dipalmitoyl phosphatidylcholine (DPPC) lipid bilayer using two different sampling conditions and two different approaches for the treatment of long-range electrostatic interactions. Specifically, sampling was carried out either by using the statistical ensembles NVT or NPT (constant number of atoms, a pressure of 1 atm in all directions and fixed temperature), and the electrostatic interactions were treated either by using a twin-cutoff, or the particle mesh Ewald summation method (PME). The results of the present study suggest that the use of the NPT ensemble in combination with the PME method provide more realistic simulations. The use of NPT during the equilibration avoids the need of an a priori estimation of the box dimensions, giving the correct area per lipid. However, once the system is equilibrated, the simulations are irrespective of the sampling conditions used. The use of an electrostatic cutoff induces artifacts on both lipid thickness and the ion distribution, but has no direct effect on the protein and water molecules.