N-terminal and internal sequence determination of microgram amounts of proteins separated by isoelectric focusing in immobilized pH gradients

Isolation of microgram amounts of proteins and submicrogram quantities of peptides in a form suitable for sequence analysis is a key step in high sensitivity protein sequencing technology. Recently, methods have been developed which allow the direct, high yield, recovery of microgram amounts of sequenceable proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis. In the present publication, we describe an extension of these methods to obtain N-terminal or internal sequence information from proteins separated by isoelectric focusing in polyacrylamide gels containing immobilized pH gradients. For N-terminal sequence analysis, separated proteins were electrophoretically transferred (electroblotted) onto chemically-modified glass fiber sheets, a support compatible with Edman degradation chemistry. Transferred protein bands were detected on the support, cut out and directly inserted into the cartridge of a gas-phase protein sequenator. For internal sequence analysis, separated proteins were electrophoretically transferred onto nitrocellulose. Protein bands were detected by staining, cut out and the proteins subjected to enzymatic digestion directly on the support. The resulting cleavage fragments (peptides) were released into the supernatant where they were recovered and separated by narrow-bore reversed-phase high performance liquid chromatography for sequence analysis. The potential of this methodology is illustrated by the comparative peptide mapping of isoforms of bovine carbonic anhydrase.     

Aquachlorobis(4,4′,4′′‐tri‐tert‐butyl‐2,2′;6′,2′′‐terpyridine‐κ3N)terbium(III) diperchlorate ethanol disolvate

In the title compound, [TbCl(C₂₇H₃₅N₃)₂(H₂O)](ClO₄)₂·2C₂H₆O, the Tb^III ion has a coordination number of eight, composed of two tridentate substituted‐ter­pyridine ligands, a water mol­ecule and a bound Cl^? anion. The first coordination shell can be described as a distorted bicapped trigonal prism. The dihedral angles between pyridine rings belonging to the same tpy ligand range from 5.2 (5) to 16.8 (5)°.     

Anodic stripping coulometry at a thin-film mercury electrode

Primary coulometric versions of d.c. anodic stripping voltammetry, performed in a microcell at a mercury-plated rotating glassy carbon electrode, are applied to the determination of cadmium and lead. With exhaustive preelectrolysis of the sample solution, 5–100 ng of the metal can be determined from the charge contained in the stripping signal; precision and accuracy are in the 5% range. An extrapolation procedure that requires only partial metal deposition is evaluated.     

Influence of structural changes on the ion selectivities of magnesium ionophores based on malonic acid diamides

In an investigation of octamethylene bis(malonic acid diamides) and their selectivities for magnesium, it was found that presence of secondary amides within the particular ionophore played a considerable role in the enhancement of magnesium selectivity. Similar effects in other ionophores, i.e. tris(malonic acid diamides), were thus systematically looked at with the help of selectivity measurements with the hope of optimizing the number of secondary and tertiary amides so as to improve the magnesium selectivity. The syntheses of several investigated tris(malonic acid diamide) isologues are equally reported.Deceased in November 1992     

Quantitative in vitro kinase reaction as a guide for phosphoprotein analysis by mass spectrometry

A simple calculation using the radioactive decay of (32)P incorporated into a protein during in vitro kinase reactions is described that allows the overall stoichiometry of phosphorylation for the substrate protein or peptide to be calculated. Prior to using techniques such as diagnostic ion scanning to identify the molecular weight of an unknown phosphopeptide in a complex mixture followed by tandem mass spectrometry (MS/MS) to locate the phosphorylated residue within the phosphopeptide, such calculations are predictive of the chances for successful characterization by these methods. An example of estimating the stoichiometry of peptide phosphorylation will be presented along with calculations that predict when adequate phosphopeptide is present in any given spot on the thin-layer chromatography (TLC) plates used for two-dimensional phosphopeptide (2DPP) mapping to allow extraction and complete characterization by MS/MS.     

Sequential superparamagnetic clustering for unbiased classification of high-dimensional chemical data

For the clustering of chemical structures that are described by the Similog, ISIS count, and ISIS binary fingerprints, we propose a sequential superparamagnetic clustering approach. To appropriately handle nonbinary feature keys, we introduce an extension of the binary Tanimoto similarity measure. In our applications, data sets composed of structures from seven chemically distinct compound classes are evaluated and correctly clustered. The comparison, with results from leading methods, indicates the superiority of our sequential superparamagnetic clustering approach.     

Fully Entangled Quantum States in C^{N^2 } and Bell Measurement

Entangled quantum states are an important component of quantum computing techniques such as quantum error-correction, dense coding, and quantum teleportation. We describe how to generate fully entangled states in the Hilbert space C ^N C ^N starting from a unitary matrix and show that they form an orthonormal basis in this space.     

A comprehensive characterization of the T-cell antigen receptor complex composition by microcapillary liquid chromatography-tandem mass spectrometry

It has become apparent that many intracellular signaling processes involve the dynamic reorganization of cellular proteins into complex signaling assemblies that have a specific subunit composition, function, and subcellular location. Since the elements of such assemblies interact physically, multiprotein signaling complexes can be isolated and analyzed. Recent technical advances in highly sensitive protein identification by electrospray-tandem mass spectrometry have dramatically increased the sensitivity with which such analyses can be performed. The T-cell antigen receptor (TCR) is an oligomeric transmembrane protein complex that is essential to T-cell recognition and function. The extracellular protein domains are responsible for ligand binding while intracellular domains generate and transduce signals in response to specific receptor-ligand interactions. We used microbore capillary chromatography-tandem mass spectrometry to investigate the composition of the TCR protein complex isolated from resting and activated cells of the murine T-cell line CD11.3. We identified all the previously known subunits of the TCR/CD3 complex as well as proteins previously not known to associate with the TCR. The catalytic activities of some of these proteins could potentially be used to interfere pharmacologically with TCR signaling.     

Phase transitions in experimental systems

Scaling functions of the support and of the measure have been used to characterize the scaling behavior of a dynamical system. While scaling functions for the scaling of the measure, ƒ(), have been calculated for a number of experimental systems, examples of scaling functions φ(λ) for the scaling of the support are difficult to obtain. In this contribution, we report on a phase-transition-like effect of an experimental p-doped germanium semiconductor sample. It is found that the results obtained from the dynamical scaling function agree with those obtained by Horita et al. from model maps, indicating that scaling functions for the scaling of the support are a powerful method of characterizing experimental dynamical systems.     

Conjugative Stabilization in an “Antiaromatic” System: The Conformational Mobility of 1,5-Bisdehydro[12]annulene

The isodynamical interconversion between the quasiplanar equilibrium conformers 1b and 1c of 1,5-bisdehydro[12]annulene requires a free energy of activation ΔG^# = 4.5 ± 0.2 kcal/mol (120 K) which was determined by line shape analysis of its temperature-dependent NMR. spectrum. Force field calculations indicate that this barrier reflects the energy needed to disrupt the cyclic π-conjugation in going to the nonplanar transition state 1a .