Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis

To increase the number of proteins detectable by two-dimensional electrophoresis (2-DE) in plants, we present a new procedure for extracting total proteins from plant tissue. This method avoids any loss of proteins in the course of sample preparation and results in two different fractions, one comprising mainly the cytoplasmatic proteins, the other one containing predominantly structure bond proteins. 2-DE patterns obtained from these two fractions show that the total number of different protein spots detected exceeds the degree of resolution commonly reported for plant proteins threefold.     

Matrix-assisted laser desorption/ionization mass spectrometry with additives to 2,5-dihydroxybenzoic acid

Selected benzoic acid derivatives and related substances were used as additives to 2,5-dihydroxybenzoic acid (2,5DHB) and the performance of the mixtures in matrix-assisted laser desorption/ionization mass spectrometry was investigated. Using benzoic acid derivatives substituted at position 2 and/or 5 or related substances as a co-matrix in the 1–10% range with 2,5DHB results in improved ion yields and signal-to-noise ratio of analyte molecules, especially for the high-mass range. The enhanced performance is prominent for 2-hydroxy-5-methoxybenzoic acid and exists for both proteins and oligosaccharides. It is suggested that the improvement is caused by a disorder in the 2,5DHB crystal lattice allowing ‘softer’ desorption. Charge transfer from matrix ions to additive molecules at the expense of analyte ionization gives a simple explanation for the deteriorating effects of some tested additives.     

Pseudopolymorphs in Industrial Use

The common property of organic and inorganic solids of forming pseudopolymorphs are of very high importance in chemical and pharmaceutical industry. Depending on the solvent some compounds form hydrates (with water) or solvates (with other solvents) in different stoichiometric proportions. Pseudopolymorphs exhibit different physical properties like density, habitus, stability, dissolution rate or bioavailability as known for polymorphs. Phase transformations, existing between the different phases, including the anhydrous or nonsolvate form, vary e.g. with temperature, pressure, relative humidity and solvent. If these process parameters are changed, phase transformations cannot be excluded, either prefered or surprising. In a common project industrial relevant properties of pseudopolymorphs are elucidated on a scientific basis.     

Synthesis and Structures of Homoleptic Methyl‐ and Phenylthiomethylaluminium Complexes [Al(CH2SR)3](R = Me,Ph)

Sulfur‐substituted methylmercury compounds [Hg(CH₂SR)₂]( 1a, R = Me; 1b, R = Ph ) react with aluminium amalgam in refluxing toluene with transmetallation to give homoleptic tris(thiomethyl)aluminium complexes [Al(CH₂SR)₃]( 2a, R = Me; 2b, R = Ph ) (degree of conversion: >80%, isolated yields: 2a 63%, 2b 41%). Their identities were confirmed by NMR spectros‐copy (¹H, ¹³C) and X‐ray crystal structure analyses. In crystals of compound 2a the aluminium atoms possess a trigonal‐bipyramidal arrangement with the coordination polyhedron defined by three carbon and two sulfur atoms. Two of the three CH₂SMe ligands are bridging ligands (μ‐η²; 1kC:2kS), the third one is terminal bound (η¹; kC). The structure is polymeric. Crystals are threaded by helical chains built up of six‐membered Al₂C₂S₂ rings. Crystals of 2b are built up of centrosymmetrical dimers with six‐membered Al₂C₂S₂ rings having bridging CH₂SPh ligands (μ‐η²; 1kC:2kS). On each Al atom two terminal (η¹; kC)CH₂SPh ligands are bound. They exhibit quite different Al‐C‐S angles (116.7(4) and 106.5(3)?). Similar values (114.32115.7? and 109.52109.9?) were found in ab initio calculations of model compounds [{Al(CH₂SR)₃}₂]( 3a, R=H; 3b, R=Me; 3c, R=CH=CH₂ ). A conformational energy diagram for rotation of one of the terminal CH₂SH ligand in the parent compound 3a around the Al‐C bond is discussed in terms of repulsive interactions of lone electron pairs of sulfur atoms.     

Investigations of the metastable decay of DNA under ultraviolet matrix-assisted laser desorption/ionization conditions with post-source-decay analysis and hydrogen/deuterium exchange

The fragmentation of positive ions of DNA under the conditions of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was investigated by post-source decay (PSD) analysis and hydrogen/deuterium (H/D) exchange. Spectra of five different synthetic 4mer oligonucleotides were recorded. As a main result the hypothesis was confirmed that for these ions all fragment ions result from processes, initiated by protonation/deuteration of a suitable base followed by a loss of this base as a neutral or ion and further backbone cleavages. The three bases adenine, guanine, and cytosine all exhibit comparable lability for fragmentation. The spectra show evidence for an interaction of the adenine base with the phosphate backbone. Signals of fragments containing TT- and CT-cycloadducts were observed in the spectra.     

Generation of minimal protein identifiers of proteins from two-dimensional gels and recombinant proteins

We describe the technical feasibility and methodology to characterize a protein by a minimal set of structural information generated by matrix assisted laser desorption/ionization (MALDI)-mass spectrometry, termed a "minimal protein Identifier" (MPI). MPIs can be determined for proteins from two-dimensional gels and recombinant proteins and can be used to compare and identify proteins from these sources.     

Large-gel two-dimensional electrophoresis-matrix assisted laser desorption/ionization-time of flight-mass spectrometry: an analytical challenge for studying complex protein mixtures

The large-gel two-dimensional electrophoresis (2-DE) technique, developed by Klose and co-workers over the past 25 years, provides the resolving power necessary to separate crude proteome extracts of higher eukaryotes. Matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) provides the sample throughput necessary to identify thousands of different protein species in an adequate time period. Spot excision, in situ proteolysis, and extraction of the cleavage products from the gel matrix, peptide purification and concentration as well as the mass spectrometric sample preparation are the crucial steps that interface the two analytical techniques. Today, these routines and not the mass spectrometric instrumentation determine how many protein digests can be analyzed per day per instrument. The present paper focuses on this analytical interface and reports on an integrated protocol and technology developed in our laboratory. Automated identification of proteins in sequence databases by mass spectrometric peptide mapping requires a powerful search engine that makes full use of the information contained in the experimental data, and scores the search results accordingly. This challenge is heading a second part of the paper.     

On the Reactivity of Platina‐β‐diketones – Synthesis and Characterization of Acylplatinum(II) Complexes

The platina‐β‐diketones [Pt₂{(COR)₂H}₂(μ‐Cl)₂] ( 1 , R = Me a , Et b ) react with phosphines L in a molar ratio of 1 : 4 through cleavage of acetaldehyde to give acylplatinum(II) complexes trans‐[Pt(COR)Cl(L)₂] ( 2 ) (R/L = Me/P(p‐FC₆H₄)₃ a , Me/P(p‐CH₂=CHC₆H₄)Ph₂ b , Me/P(n‐Bu)₃ c , Et/P(p‐MeOC₆H₄)₃ d ). 1 a reacts with Ph₂As(CH₂)₂PPh₂ (dadpe) in a molar ratio of 1 : 2 through cleavage of acetaldehyde yielding [Pt(COMe)Cl(dadpe)] ( 3 a ) (configuration index: SP‐4‐4) and [Pt(COMe)Cl(dadpe)] (configuration index: SP‐4‐2) ( 3 b ) in a ratio of about 9 : 1. All acyl complexes were characterized by ¹H, ¹³C and ³¹P NMR spectroscopy. The molecular structures of 2 a and 3 a were determined by single‐crystal X‐ray diffraction. The geometries at the platinum centers are close to square planar. In both complexes the plane of the acyl ligand is nearly perpendicular to the plane of the complex (88(2)° 2 a , 81.2(5)° 3 a ).     

Matrix-assisted laser desorption/ionization mass spectrometry of nucleic acids with wavelengths in the ultraviolet and infrared.

A number of different matrices have been tested and compared for ultraviolet and infrared (UV and IR) matrix-assisted laser desorption/ionization (MALDI) of oligodeoxyribonucleotides and mixtures thereof, as well as ribonucleic acids (tRNA from yeast and rRNA from E. coli). A new technique for removing alkali cations from nucleic acid samples during sample preparation on the sample support is demonstrated. The amount of oligonucleotide sample consumed during a typical measurement in IR-MALDI-MS was determined.