Protein mapping by two-dimensional high performance liquid chromatography

Current developments in drug discovery in the pharmaceutical industry require highly efficient analytical systems for protein mapping providing high resolution, robustness, sensitivity, reproducibility and a high throughput of samples. The potential of two-dimensional (2D) HPLC as a complementary method to 2D-gel electrophoresis is investigated, especially in view of speed and repeatability. The method will be applied for proteins of a molecular mass <20 000 which are not well resolved in 2D-gel electrophoresis. The 2D-HPLC system described in this work consisted of anion- or cation-exchange chromatography in the first dimension and reversed-phase chromatography in the second dimension. We used a comprehensive two-dimensional approach based on different separation speeds. In the first dimension 2.5 microm polymeric beads bonded with diethylaminoethyl and sulfonic acid groups, respectively, were applied as ion exchangers and operated at a flow-rate of 1 ml/min. To achieve very high-speed and high-resolution separations in the second dimension, short columns of 14 x 4.6 mm I.D. with 1.5 microm n-octadecyl bonded, non-porous silica packings were chosen and operated at a flow-rate of 2.5 ml/min. Two reversed-phase columns were used in parallel in the second dimension. The analyte fractions from the ion-exchange column were transferred alternatively to one of the two reversed-phase columns using a 10-port switching valve. The analytes were deposited in an on-column focusing mode on top of one column while the analytes on the second column were eluted. Proteins, which were not completely resolved in the first dimension can, in most cases, be baseline-separated in the second dimension. The total value of peak capacity was calculated to 600. Fully unattended overnight runs for repeatability studies proved the applicability of the system. The values for the relative standard deviation (RSD) of the retention times of proteins were less than 1% (n = 15), while the RSDs of the peak areas were less than 15% (n = 15) on average. The limit of detection was 300 ng of protein on average and decreased to 50 ng for ovalbumin. The 2D-HPLC system offered high-resolution protein separations with a total analysis time of less than 20 min, equivalent to the run time of the first dimension.     

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.     

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.