Lech-Gustav Milroy,† Tom N. Grossmann,‡,§ Sven Hennig,‡ Luc Brunsveld,† and Christian Ottmann*,† †Laboratory of Chemical Biology and Institute of Complex Molecular Systems, Department of Biomedical Engineering, Technische Universiteit Eindhoven, Den Dolech 2, 5612 AZ Eindhoven, The Netherlands ‡Chemical Genomics Centre of the Max Planck Society, Otto-Hahn Straße 15, 44227 Dortmund, Germany Department of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany

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Modulators of Protein-Protein Interactions

Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed.

https://www.mdpi.com/1422-0067/16/2/3537/pdf

Proteomic Challenges: Sample Preparation Techniques for Microgram-Quantity Protein Analysis From Biological Samples

Isothermal titration calorimetry (ITC) can provide a full thermodynamic characterization of an interaction. Its usage does not suffer from constraints of molecular size, shape or chemical constitution. Neither is there any need for chemical modification or attachment to solid support. This ease of use has made it an invaluable instrumental resource and led to its appearance in many laboratories. Despite this, the value of the thermodynamic parameterization has, only quite recently, become widely appreciated. Although our understanding of the correlation between thermodynamic data and structural details continues to be somewhat naive, a large number of publications have begun to improve the situation. In this overview of the literature for 2005, we have attempted to highlight works of interest and novelty. Furthermore, we draw attention to those works which we feel have provided a route to better analysis and increased our ability to understand the meaning of thermodynamic change on binding.

Survey of the year 2005: literature on applications of isothermal titration calorimetry.

Studies of gene–environment (G × E) interactions require effective characterization of all environmental exposures from conception to death, termed the exposome. The exposome includes environmental exposures that impact health. Improved metabolic profiling methods are needed to characterize these exposures for use in personalized medicine. In the present study, we compared the analytic capability of dual chromatography-Fourier-transform mass spectrometry (DC-FTMS) to previously used liquid chromatography-FTMS (LC-FTMS) analysis for high-throughput, top-down metabolic profiling. For DC-FTMS, we combined data from sequential LC-FTMS analyses using reverse phase (C18) chromatography and anion exchange (AE) chromatography. Each analysis was performed with electrospray ionization in the positive ion mode and detection from m/z 85 to 850. Run time for each column was 10 min with gradient elution; 10 μl extracts of plasma from humans and common marmosets were used for analysis. In comparison to analysis with the AE column alone, addition of the second LC-FTMS analysis with the C18 column increased m/z feature detection by 23–36%, yielding a total number of features up to 7,000 for individual samples. Approximately 50% of the m/z matched to known chemicals in metabolomic databases, and 23% of the m/z were common to analyses on both columns. Database matches included insecticides, herbicides, flame retardants, and plasticizers. Modularity clustering algorithms applied to MS-data showed the ability to detection clusters and ion interactions. DC-FTMS thus provides improved capability for high-performance metabolic profiling of the exposome and development of personalized medicine.

/pdf/high-performance-metabolic-profiling-with-dual-5sgz7llg7w.pdf

High-performance metabolic profiling with dual chromatography-Fourier-transform mass spectrometry (DC-FTMS) for study of the exposome.

What Protein Charging (and Supercharging) Reveal about the Mechanism of Electrospray Ionization

DMSO is the standard solvent for preparing stock solutions of compounds for drug discovery. The assay concentration of DMSO is normally 0.1% to 5% (v/v) or 14 to 715 mM. Thus, DMSO is often one of the principal additives in assay buffers. This standardization of stock solutions does not eliminate possible pitfalls associated with the effects of the DMSO-containing solutions on individual proteins. In this article, the authors want to emphasize the importance of detailed studies of these effects in the early stages of drug discovery. Two protein systems, the extracellular soluble domain of the human growth hormone receptor (hGHbp) and the phosphatase domain of PFKFB1 (BPase), were used for the study on effects of DMSO on protein stability, protein aggregation, and binding of drug compounds. The study revealed significant differences in the proteins' behavior in the presence and absence of low amounts of DMSO. The addition of DMSO resulted in destabilization of the proteins investigated and also changed the apparent binding property of 1 protein. The authors have also shown that low DMSO concentrations influence the ionization process in electrospray ionization mass spectrometry (ESI-MS).

https://journals.sagepub.com/doi/pdf/10.1177/1087057105284218

DMSO-Related Effects in Protein Characterization

The development of a continuous isothermal titration calorimetric method for equilibrium studies.

The TWEAK-Fn14 pathway is upregulated in models of inflammation, autoimmune diseases, and cancer. Both TWEAK and Fn14 show increased expression also in the CNS in response to different stimuli, particularly astrocytes, microglia, and neurons, leading to activation of NF-κB and release of proinflammatory cytokines. Although neutralizing antibodies against these proteins have been shown to have therapeutic efficacy in animal models of inflammation, no small-molecule therapeutics are yet available. Here, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF)-based screening assay together with several counterassays for the identification of small-molecule inhibitors of this protein-protein interaction. Recombinant HIS-TWEAK and Fn14-Fc proteins as well as FLAG-TWEAK and Fn14-FLAG proteins and an anti-Fn14 antibody were used to establish and validate these assays and to screen a library of 60 000 compounds. Two HTRF counterassays with unrelated proteins in the same assay format, an antiaggregation assay and a redox assay, were applied to filter out potential false-positive compounds. The novel assay and associated screening cascade should be useful for the discovery of small-molecule inhibitors of the TWEAK-Fn14 protein interaction.

https://journals.sagepub.com/doi/pdf/10.1177/1087057112447873

Natalia Markova

Papers

DMSO-Related Effects in Protein Characterization

The development of a continuous isothermal titration calorimetric method for equilibrium studies.

A homogeneous HTRF assay for the identification of inhibitors of the TWEAK-Fn14 protein interaction.