Insight into Signal Response of Protein Ions in Native ESI-MS from the Analysis of Model Mixtures of Covalently Linked Protein Oligomers

Native ESI-MS is increasingly used for quantitative analysis of biomolecular interactions. In such analyses, peak intensity ratios measured in mass spectra are treated as abundance ratios of the respective molecules in solution. While signal intensities of similar-size analytes, such as a protein and its complex with a small molecule, can be directly compared, significant distortions of the peak ratio due to unequal signal response of analytes impede the application of this approach for large oligomeric biomolecular complexes. We use a model system based on concatenated maltose binding protein units (MBPn, n = 1, 2, 3) to systematically study the behavior of protein mixtures in ESI-MS. The MBP concatamers differ from each other only by their mass while the chemical composition and other properties remain identical. We used native ESI-MS to analyze model mixtures of MBP oligomers, including equimolar mixtures of two proteins, as well as binary mixtures containing different fractions of the individual components. Pronounced deviation from a linear dependence of the signal intensity with concentration was observed for all binary mixtures investigated. While equimolar mixtures showed linear signal dependence at low concentrations, distinct ion suppression was observed above 20 μM. We systematically studied factors that are most often used in the literature to explain the origin of suppression effects. Implications of this effect for quantifying protein–protein binding affinity by native ESI-MS are discussed in general and demonstrated for an example of an anti-MBP antibody with its ligand, MBP.

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Analysis of aquatic‐phase natural organic matter by optimized LDI‐MS method

The composition and physiochemical properties of aquatic‐phase natural organic matter (NOM) are most important problems for both environmental studies and water industry. Laser desorption/ionization (LDI) mass spectrometry facilitated successful examinations of NOM, as humic and fulvic acids in NOM are readily ionized by the nitrogen laser. In this study, hydrophobic NOMs (HPO NOMs) from river, reservoir and waste water were characterized by this technique. The effect of analytical variables like concentration, solvent composition and laser energy was investigated. The exact masses of small molecular NOM moieties in the range of 200–1200 m/z were determined in reflectron mode. In addition, spectra of post‐source‐decay experiments in this range showed that some compounds from different natural NOMs had the same fragmental ions. In the large mass range of 1200–15 000 Da, macromolecules and their aggregates were found in HPO NOMs from natural waters. Highly humic HPO exhibited mass peaks larger than 8000 Da. On the other hand, the waste water and reservoir water mainly had relatively smaller molecules of about 2000 Da. The LDI‐MS measurements indicated that highly humic river waters were able to form large aggregates and membrane foulants, while the HPO NOMs from waste water and reservoir water were unlikely to form large aggregates. Copyright © 2014 John Wiley & Sons, Ltd.     

Electron Transfer in Peptides: On the Formation of Silver Nanoparticles

Some microorganisms perform anaerobic mineral respiration by reducing metal ions to metal nanoparticles, using peptide aggregates as medium for electron transfer (ET). Such a reaction type is investigated here with model peptides and silver as the metal. Surprisingly, Ag⁺ ions bound by peptides with histidine as the Ag⁺‐binding amino acid and tyrosine as photoinducible electron donor cannot be reduced to Ag nanoparticles (AgNPs) under ET conditions because the peptide prevents the aggregation of Ag atoms to form AgNPs. Only in the presence of chloride ions, which generate AgCl microcrystals in the peptide matrix, does the synthesis of AgNPs occur. The reaction starts with the formation of 100 nm Ag@AgCl/peptide nanocomposites which are cleaved into 15 nm AgNPs. This defined transformation from large nanoparticles into small ones is in contrast to the usually observed Ostwald ripening processes and can be followed in detail by studying time‐resolved UV/Vis spectra which exhibit an isosbestic point.     

Structural Design Principle of Small‐Molecule Organic Semiconductors for Metal‐Free,Visible‐Light‐Promoted Photocatalysis

Herein, we report on the structural design principle of small‐molecule organic semiconductors as metal‐free, pure organic and visible light‐active photocatalysts. Two series of electron‐donor and acceptor‐type organic semiconductor molecules were synthesized to meet crucial requirements, such as 1) absorption range in the visible region, 2) sufficient photoredox potential, and 3) long lifetime of photogenerated excitons. The photocatalytic activity was demonstrated in the intermolecular C?H functionalization of electron‐rich heteroaromates with malonate derivatives. A mechanistic study of the light‐induced electron transport between the organic photocatalyst, substrate, and the sacrificial agent are described. With their tunable absorption range and defined energy‐band structure, the small‐molecule organic semiconductors could offer a new class of metal‐free and visible light‐active photocatalysts for chemical reactions.     

Acoustic Radiation Force Impulse-Imaging for the evaluation of the thyroid gland: a limited patient feasibility study

Purpose

Real-time tissue elastography, a qualitative elastography method, has shown promising results in the diagnostic work up of thyroid nodules. However, to our knowledge no study has evaluated a quantitative elastography method in the thyroid gland. The present study is a feasibility study evaluating Acoustic Radiation Force Impulse-Imaging, a novel quantitative elastography method in the thyroid gland.

Methods

ARFI-imaging involves the mechanical excitation of tissue using short-duration acoustic pulses to generate localized displacements in tissue. The displacements induce a lateral shear-wave propagation which is tracked using multiple laterally positioned ultrasound “tracking“ beams. Inclusion criteria were: thyroid nodules ?1 cm, non-functioning or hypo-functioning on radionuclide scanning, and cytological/histological assessment of thyroid nodule as reference method. All patients received conventional ultrasound, and examination of the thyroid gland including Power Doppler Ultrasound using a 9 MHz linear transducer, in addition real-time elastography (RTE) was performed at 9 MHz frequency and ARFI-imaging was performed at 4 MHz using Siemens (ACUSON S2000) B-mode-ARFI combination transducer.

Results

Sixty nodules in 55 patients were analyzed. Three nodules were papillary carcinoma. The stiffer the tissue the faster the shear wave propagates. The results obtained indicated that the shear wave velocity in thyroid lobes ranged between 0.5 and 4.9 m/s. The median velocity of ARFI-imaging in the healthy nodule-free thyroid gland, as well as in benign and malignant thyroid nodules was 1.98 m/s (range: 1.20-3.63 m/s), 2.02 m/s (range: 0.92-3.97 m/s), and 4.30 m/s (range: 2.40-4.50 m/s), respectively. While no significant difference in median velocity was found between healthy thyroid tissue and benign thyroid nodules, a significant difference was found between malignant thyroid nodules on the one hand and healthy thyroid tissue (p = 0.018) or benign thyroid nodules (p = 0.014) on the other hand. Specificity of ARFI-imaging for the differentiation of benign and malignant thyroid nodules was comparable with RTE (91-95%).

Conclusions

ARFI can be performed in the thyroid tissue with reliable results.