Günter Allmaier

Transferrin binding and transferrin-mediated cellular uptake of the ruthenium coordination compound KP1019, studied by means of AAS, ESI-MS and CD spectroscopy

Indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) shows particular promise as an antitumour agent against colorectal cancer. It is known that KP1019 reacts with human serum proteins, whereby the major amount binds to albumin (present in large excess) and a smaller amount to transferrin. It has been hypothesised that transferrin-mediated uptake by transferrin receptor expressing tumour cells may in part explain the apparent tumour selectivity of this compound. Circular dichroism spectroscopy and electrospray ionisation mass spectrometry studies demonstrate that two equivalents of KP1019 bind specifically to human apotransferrin, while additional amounts of the ruthenium complex bind unspecifically. Uptake studies in the transferrin receptor-expressing human colon carcinoma cell line SW480 revealed a higher cellular accumulation of KP1019 in comparison to a KP1019-transferrin adduct (2∶1), while the uptake of a KP1019–Fe(III)-transferrin conjugate (1∶0.3∶1) significantly exceeded that of KP1019, suggesting that iron binding is necessary to obtain a protein conformation which favours recognition by the transferrin receptors on the cell surface. Our study showed that KP1019 is transported into the cell by both transferrin-independent and transferrin-dependent mechanisms. Transferrin-mediated uptake is more efficient when transferrin is saturated with iron to a physiological degree (∼30%). Cell fractionation experiments demonstrated that after a 2 h treatment of human colon cancer cells with 10 µM KP1019 on average 55% of the intracellular ruthenium is located in the cellular nucleus, while 45% remain in the cytosol and other cellular components.

Epl1, the major secreted protein of Hypocrea atroviridis on glucose, is a member of a strongly conserved protein family comprising plant defense response elicitors.

We used a proteomic approach to identify constitutively formed extracellular proteins of Hypocrea atroviridis (Trichoderma atroviride), a known biocontrol agent. The fungus was cultivated on glucose and the secretome was examined by two‐dimensional gel electrophoresis. The two predominant spots were identified by MALDI MS utilizing peptide mass fingerprints and amino acid sequence tags obtained by postsource decay and/or high‐energy collision‐induced dissociation (MS/MS) experiments, and turned out to be the same protein (12 629 Da as determined with MS, pI 5.5–5.7), probably representing the monomer and the dimer. The corresponding gene was subsequently cloned from H. atroviridis and named epl1 (eliciting plant response‐like), because it encodes a protein that exhibits high similarity to the cerato‐platanin family, which comprises proteins such as cerato‐platanin from Ceratocystis fimbriata f. sp. platani and Snodprot1 of Phaeosphaeria nodorum, which have been reported to be involved in plant pathogenesis and elicitation of plant defense responses. Additionally, based on the similarity of the N‐terminus to that of H. atroviridis Epl1, we conclude that a previously identified 18 kDa plant response elicitor isolated from T. virens is an ortholog of epl1. Our results showed that epl1 transcript was present under all growth conditions tested, which included the carbon sources glucose, glycerol, l‐arabinose, d‐xylose, colloidal chitin and cell walls of the plant pathogen Rhizoctonia solani, and also plate confrontation assays with R. solani. Epl1 transcript could even be detected under osmotic stress, and carbon and nitrogen starvation.

Divergent Coupling of Alcohols and Amines Catalyzed by Isoelectronic Hydride Mn(I) and Fe(II) PNP Pincer Complexes.

Herein, we describe an efficient coupling of alcohols and amines catalyzed by well-defined isoelectronic hydride Mn(I) and Fe(II) complexes, which are stabilized by a PNP ligand based on the 2,6-diaminopyridine scaffold. This reaction is an environmentally benign process implementing inexpensive, earth-abundant non-precious metal catalysts, and is based on the acceptorless alcohol dehydrogenation concept. A range of alcohols and amines including both aromatic and aliphatic substrates were efficiently converted in good to excellent isolated yields. Although in the case of Mn selectively imines were obtained, with Fe-exclusively monoalkylated amines were formed. These reactions proceed under base-free conditions and required the addition of molecular sieves.

Evaluation of matrix-assisted laser desorption/ionization (MALDI) preparation techniques for surface characterization of intact Fusarium spores by MALDI linear time-of-flight mass spectrometry

Unambiguous identification of mycotoxin-producing fungal species as Fusarium is of great relevance to agriculture and the food-producing industry as well as in medicine. Protein profiles of intact fungal spores, such as Penicillium, Aspergillus and Trichoderma, derived from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were shown to provide a rapid and straightforward method for species identification and characterization. In this study, we applied this approach to five different Fusarium spp. strains which are known to affect the growth of different grain plants. To obtain a suitable MALDI matrix system and sample preparation method, thin-layer, dried-droplet and sandwich methods and several MALDI matrices, namely CHCA, DHB, FA, SA and THAP dissolved in various solvent mixtures (organic solvents such as ACN, MeOH, EtOH and iPrOH and for the aqueous phase water and 0.1% TFA), were evaluated in terms of mass spectrometric pattern and signal intensities. The most significant peptide/protein profiles were obtained with 10 mg ferulic acid (FA) in 1 mL ACN/0.1% TFA (7:3, v/v) used as matrix system. Mixing the spores with the matrix solution directly on the MALDI target (dried-droplet technique) resulted in an evenly distributed spores/matrix crystal layer, yielding highly reproducible peptide/protein profiles from the spore surfaces. Numerous abundant ions throughout the investigated m/z range (m/z 1500-15 000) could be detected. Differences in the obtained mass spectral patterns allowed the differentiation of spores of various Fusarium species.

The Renaissance of High-Energy CID for Structural Elucidation of Complex Lipids: MALDI-TOF/RTOF-MS of Alkali Cationized Triacylglycerols

Triacylglycerols were analyzed as cationized species (Li+, Na+, K+) by high-energy CID at 20 keV collisions utilizing MALDI-TOF/RTOF mass spectrometry. Precursor ions, based on [M+Li]+-adduct ions exhibited incomplete fragmentation in the high and low m/z region whereas [M+K]+-adducts did not show useful fragmentation. Only sodiated precursor ions yielded product ion spectra with structurally diagnostic product ions across the whole m/z range. The high m/z region of the CID spectra is dominated by abundant charge-remote fragmentation of the fatty acid substituents. In favorable cases also positions of double bonds or of hydroxy groups of the fatty acid alkyl chains could be determined. A-type product ions represent the end products of these charge-remote fragmentations. B- and C-type product ions yield the fatty acid composition of individual triacylglycerol species based on loss of either one neutral fatty acid or one sodium carboxylate residue, respectively. Product ions allowing fatty acid substituent positional determination were present in the low m/z range enabling identification of either the sn-1/sn-3 substituents (E-, F-, and G-type ions) or the sn-2 substituent (J-type ion). These findings were demonstrated with synthetic triacylglycerols and plant oils such as cocoa butter, olive oil, and castor bean oil. Typical features of 20 keV CID spectra of sodiated triacylglycerols obtained by MALDI-TOF/RTOF MS were an even distribution of product ions over the entire m/z range and a mass accuracy of ±0.1 to 0.2 u. One limitation of the application of this technique is mainly the insufficient precursor ion gating after MS1 (gating window at 4 u) of species separated by 2 u.

Efficient Hydrogenation of Ketones and Aldehydes Catalyzed by Well-Defined Iron(II) PNP Pincer Complexes: Evidence for an Insertion Mechanism

We have prepared and structurally characterized a new class of Fe(II) PNP pincer hydride complexes [Fe(PNP-iPr)(H)(CO)(L)]n (L = Br–, CH3CN, pyridine, PMe3, SCN–, CO, BH4–; n = 0, +1) based on the 2,6-diaminopyridine scaffold where the PiPr2 moieties of the PNP ligand are connected to the pyridine ring via NH and/or NMe spacers. Complexes [Fe(PNP-iPr)(H)(CO)(L)]n with labile ligands (L = Br–, CH3CN, BH4–) and NH spacers are efficient catalysts for the hydrogenation of both ketones and aldehydes to alcohols under mild conditions, while those containing inert ligands (L = pyridine, PMe3, SCN–, CO) are catalytically inactive. Interestingly, complex [Fe(PNPMe-iPr)(H)(CO)(Br)], featuring NMe spacers, is an efficient catalyst for the chemoselective hydrogenation of aldehydes. The first type of complexes involves deprotonation of the PNP ligand as well as heterolytic dihydrogen cleavage via metal-alkoxide cooperation, but no reversible aromatization/deprotonation of the PNP ligand. In the case of the N-methylated complex the mechanism remains unclear, but obviously does not allow bifunctional activation of dihydrogen. The experimental results complemented by DFT calculations strongly support an insertion of the C=O bond of the carbonyl compound into the Fe–H bond.

Biological Variation of the Platelet Proteome in the Elderly Population and Its Implication for Biomarker Research

Knowledge about the extent of total variation experienced between samples from different individuals is of great importance for the design of not only proteomics but every clinical study. This variation defines the smallest statistically significant detectable signal difference when comparing two groups of individuals. We isolated platelets from 20 healthy human volunteers aged 56–100 years because this age group is most commonly encountered in the clinics. We determined the technical and total variation experienced in a proteome analysis using two-dimensional DIGE with IPGs in the pI ranges 4–7 and 6–9. Only spots that were reproducibly detectable in at least 90% of all gels (n = 908) were included in the study. All spots had a similar technical variation with a median coefficient of variation (cv) of about 7%. In contrast, spots showed a more diverse total variation between individuals with a surprisingly low median cv of only 18%. Because most known biomarkers show an effect size in a 1–2-fold range of their cv, any future clinical proteomics study with platelets will require an analytical method that is able to detect such small quantitative differences. In addition, we calculated the minimal number of samples (sample size) needed to detect given protein expression differences with statistical significance.

Nano ES GEMMA and PDMA, new tools for the analysis of nanobioparticles—Protein complexes, lipoparticles, and viruses

Differential mobility analysis (DMA) is a technique suited for size analysis as well as preparative collection of airborne nanosized airborne particles. In the recent decade, the analysis of intact viruses, proteins, DNA fragments, polymers, and inorganic nanoparticles was possible when combining this method with a nano-electrospray charge-reduction source for producing aerosols from a sample solution/suspensions. Mass analysis of high molecular weight noncovalent complexes is also possible with this methodology due to the linear correlation of the electrophoretic mobility diameter and the molecular mass. In this work, we present the analysis (size and molecular mass) of high molecular weight multimers (noncovalent functional homocomplex) of Jack bean urease in a mass range from 275 kDa up to 2.5 MDa, with mainly present tri- and hexamers but also higher oligomers of the 91 kDa monomer subunit. In a second experiment, the size analysis of intact very-low-density (∼35 nm), low-density (∼22 nm) and high-density lipoparticles (∼10 nm), which are heterocomplexes consisting of cholesterol, lipids, and proteins in different ratios, is presented. Results from mobility analysis were in excellent agreement with particle diameters found in literature. The last presented experiment demonstrates size analysis of a rod-like virus and selective sampling of a selected size fraction of electrosprayed, singly-charged tobacco mosaic virus particles. Sampling and subsequent transmission electron microscopic investigations of a specific size fraction (40 nm electrophoretic mobility diameter) revealed the folding of virus particles during the electrospray and charge reduction (electrical stress) as well as solvent evaporation (mechanical stress) process, leading to an observed geometry of 150 (length) × 35 (width) nm (average cylindrical geometry of unsprayed intact virus 300 × 18 nm).

Development of a MALDI two-layer volume sample preparation technique for analysis of colored conidia spores of Fusarium by MALDI linear TOF mass spectrometry

AbstractMatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been proved to be a powerful tool for the identification and characterization of microorganisms based on their surface peptide/protein pattern. Because of the complexity of microorganisms, there are no standardized protocols to acquire reproducible peptide/protein profiles for a broad range of microorganisms and for fungi in particular. Small variations during MALDI MS sample preparation affect the quality of mass spectra quite often. In this study, we were aiming to develop a sample preparation method for the analysis of colored, a quite often observed phenomenon, and mycotoxin-producing Fusarium conidia spores using MALDI–TOF MS. Different washing solvent systems for light- and deep-colored (from slightly orange to red-brown) conidia spores and connected sample deposition techniques were evaluated based on MS reproducibility and number and intensities of peaks. As a method of choice for generation of reproducible and characteristic MALDI–TOF mass spectra, the use of a washing process for colored Fusarium conidia spores with acetonitrile/0.5% formic acid (7/3) was found and subsequently combined with two-layer volume technique (spores/matrix (ferulic acid) solution was deposited onto a MALDI target, and after solvent evaporation, a second matrix layer was deposited). With the application of this sample preparation method, for deep-colored Fusarium species, 19 abundant molecular ions in the m/z range 2,000–10,000 were always detected with an S/N ratio of 3:1 or better. Finally this optimized sample preparation for the first time provided mass spectrometric fingerprints of strongly colored Fusarium conidia spores resulting in the possibility of differentiation of such spores at the species level. FigureIntact spore mass spectrometry - from fungal conidia spores to optimal MALDI TOF mass spectrum for species differentiation and identification.

Gas-phase electrophoretic molecular mobility analysis of size and stoichiometry of complexes of a common cold virus with antibody and soluble receptor molecules.

Attachment of a nonaggregating monoclonal antibody and of a soluble recombinant receptor molecule to the icosahedral nonenveloped human rhinovirus serotype 2 was studied with a nanoelectrospray ionization gas-phase electrophoretic molecular mobility analyzer (nESI-GEMMA). The virus mass, as determined via nESI-GEMMA, was within instrument accuracy (+/-6%) close to the theoretical value (8 x 10(6) Da) calculated from the sum of all constituents of one virus particle (60 copies of each of the four viral capsid proteins, the RNA genome, and one copy of the RNA-linked protein VpG). The formation of virus-antibody complexes of different stoichiometries (up to a mass 12.5 x 10(6) Da corresponding to 30 attached antibodies) and virus-receptor complexes (up to a mass 8.8 x 10(6) Da corresponding to 12 attached receptor molecules) was monitored. Via the volume derived from the electrophoretic mobility diameter (EMD), the stoichiometry of the HRV complexes was calculated. The accuracy of the EMD was within +/-0.5 nm, which corresponds to an accuracy of +/-4 antibodies and +/-5 receptor molecules in the respective complexes. For the first time, we here demonstrate the use of nESI-GEMMA for the analysis of the size and stoichiometry of biomolecules in high-order complexes in real time under normal pressure conditions.