Basri Gülbakan

Determination of thermodynamic and kinetic properties of biomolecules by mass spectrometry.

Over the past two decades, mass spectrometry (MS) has transformed the life sciences. The advances in understanding biomolecule structure and function by MS is progressing at an accelerated pace. MS has also largely been applied to study thermodynamic and kinetic structure of biomolecules. Herein, we highlight the recent discussions about native mass spectrometry and studies about determining stable gas phase structures, hydrogen/deuterium exchange studies about reaction kinetics and determination of binding constants of biomolecules with their ligands.

Generation of Fluorogen-Activating Designed Ankyrin Repeat Proteins (FADAs) as Versatile Sensor Tools

Fluorescent probes constitute a valuable toolbox to address a variety of biological questions and they have become irreplaceable for imaging methods. Commonly, such probes consist of fluorescent proteins or small organic fluorophores coupled to biological molecules of interest. Recently, a novel class of fluorescence-based probes, fluorogen-activating proteins (FAPs), has been reported. These binding proteins are based on antibody single-chain variable fragments and activate fluorogenic dyes, which only become fluorescent upon activation and do not fluoresce when free in solution. Here we present a novel class of fluorogen activators, termed FADAs, based on the very robust designed ankyrin repeat protein scaffold, which also readily folds in the reducing environment of the cytoplasm. The FADA generated in this study was obtained by combined selections with ribosome display and yeast surface display. It enhances the fluorescence of malachite green (MG) dyes by a factor of more than 11,000 and thus activates MG to a similar extent as FAPs based on single-chain variable fragments. As shown by structure determination and in vitro measurements, this FADA was evolved to form a homodimer for the activation of MG dyes. Exploiting the favorable properties of the designed ankyrin repeat protein scaffold, we created a FADA biosensor suitable for imaging of proteins on the cell surface, as well as in the cytosol. Moreover, based on the requirement of dimerization for strong fluorogen activation, a prototype FADA biosensor for in situ detection of a target protein and protein-protein interactions was developed. Therefore, FADAs are versatile fluorescent probes that are easily produced and suitable for diverse applications and thus extend the FAP technology.

Influence of Ammonium Acetate Concentration on Receptor–Ligand Binding Affinities Measured by Native Nano ESI-MS: A Systematic Study

Native electrospray ionization (ESI) mass spectrometry (MS) is a powerful technique for analyzing biomolecules in their native state. However, ESI-MS is incompatible with nonvolatile solution additives. Therefore, biomolecules have to be electrosprayed from a solution that differs from their purification or storage buffer, often aqueous ammonium acetate (AmAc). In this study, the effect of the ionic strength on the dissociation constants of six different noncovalent complexes, that cover interactions present in many biological systems, was investigated. Complexes were electrosprayed from 10 mM, 50 mM, 100 mM, 300 mM, and 500 mM aqueous AmAc. For all systems, it was shown that the binding affinity is significantly influenced by the ionic strength of the solution. The determined dissociation constant (Kd) was affected more than 50% when increasing the AmAc concentration. The results are interpreted in terms of altered ionic interactions induced by the solution. This work emphasizes the modulating effect of the ions on noncovalent interactions and the importance of carefully choosing the AmAc concentration for quantifying the receptor-ligand binding strengths.

DNA oligonucleotides: a model system with tunable binding strength to study monomer-dimer equilibria with electrospray ionization-mass spectrometry.

Electrospray ionization (ESI) is increasingly used to measure binding strengths, but it is not always clear whether the ESI process introduces artifacts. Here we propose a model monomer-dimer equilibrium system based on DNA oligonucleotides to systematically explore biomolecular self-association with the ESI-mass spectrometry (MS) titration method. The oligonucleotides are designed to be self-complementary and have the same chemical composition and mass, allowing for equal ionization probability, ion transmission, and detection efficiency in ESI-MS. The only difference is the binding strength, which is determined by the nucleotide sequence and can be tuned to cover a range of dissociation constant values. This experimental design allows one to focus on the impact of ESI on the chemical equilibrium and to avoid the other typical sources of variation in ESI-MS signal responses, which yields a direct comparison of samples with different binding strengths. For a set of seven model DNA oligonucleotides, the monomer-dimer binding equilibrium was probed with the ESI-MS titration method in both positive and negative ion modes. A mathematical model describing the dependence of the monomer-to-dimer peak intensity ratio on the DNA concentration was proposed and used to extract apparent Kd values and the fraction of DNA duplex that irreversibly dissociates in the gas phase. The Kd values determined via ESI-MS titration were compared to those determined in solution with isothermal titration calorimetry and equilibrium thermal denaturation methods and were found to be significantly lower. The observed discrepancy was attributed to a greater electrospray response of dimers relative to that of monomers.

Direct access to aptamer–protein complexes via MALDI-MS

We report on the direct detection of protein–aptamer complexes by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). By using optimized conditions, we were able to observe the complexes of thrombin and two different thrombin binding aptamers (TBAs) directly. We also detected the complex of PDGF-AB/BB with the specific PDGF binding aptamer (Apt-35) in a 1 : 2 stoichiometry. Detection of the complex between lysozyme and its corresponding aptamer further confirmed the capability of MALDI-MS for studying such systems. All these analyses could be performed with very low sample concentrations (1 pmol) and volumes (1–10 μL). Well-designed control experiments confirmed that the complex observation is due to specific non-covalent interactions, rather than non-specific clusters formed in the MALDI plume. The stronger thrombin–TBA29 complex showed a larger signal at the m/z of the intact complex than the weaker thrombin–TBA15 complex; the complex signal of Apt-35 and PDGF-BB was stronger in MALDI compared with that of PDGF-AB and PDGF-AA. These observations indicate that the noncovalent interaction strength in solution is reflected in the MALDI mass spectra.

Discovery of biomarkers in rare diseases: innovative approaches by predictive and personalized medicine

There are more than 8000 rare diseases (RDs) that affect >5 % of the world’s population. Many of the RDs have no effective treatment and lack of knowledge creates delayed diagnosis making management difficult. The emerging concept of the personalized medicine allows for early screening, diagnosis, and individualized treatment of human diseases. In this context, the discovery of biomarkers in RDs will be of prime importance to enable timely prevention and effective treatment. Since 80 % of RDs are of genetic origin, identification of new genes and causative mutations become valuable biomarkers. Furthermore, dynamic markers such as expressed genes, metabolites, and proteins are also very important to follow prognosis and response the therapy. Recent advances in omics technologies and their use in combination can define pathophysiological pathways that can be drug targets. Biomarker discovery and their use in diagnosis in RDs is a major pillar in RD research.

Oligonucleotide aptamers: emerging affinity probes for bioanalytical mass spectrometry and biomarker discovery

Selective isolation of biologically important molecules and their functional characterization is one of the primary goals of bioanalytical chemistry. Several different affinity tools such as antibodies, affimers, nanobodies, and DARPins have been explored to achieve these goals. In recent years, oligonucleotide based affinity tools called aptamers have become progressively attractive and the research in this area has seen an exponential increase. Aptamer probes have been explored in many different areas of bioanalytical chemistry such as electrochemical and optical biosensor development, targeted drug delivery, logic gates, DNA nanotechnology, and point of care diagnostics. However aptamers are still largely overlooked in mass spectrometry (MS) and biomarker discovery. After the completion of the human genome project, the focus has shifted towards functional genomics and to understand the living systems by deciphering the functions of proteins and metabolites. Therefore identification and functional characterization of these molecules are of outmost importance. Although identification of isolated biomolecules and analysis of simple biological mixtures using MS have become relatively simple, the power of MS gradually decreases as the complexity of biological mixtures increases. Therefore the development of selective and targeted approaches is at the forefront of mass spectrometry. Aptamers have great potential in affinity mass spectrometry to improve selectivity, specificity and throughput. Herein, bioanalytical mass spectrometry and biomarker discovery applications of aptamers will be reviewed.

Native Electrospray Ionization Mass Spectrometry Reveals Multiple Facets of Aptamer–Ligand Interactions: From Mechanism to Binding Constants

Aptamers are oligonucleotide receptors obtained through an iterative selection process from random-sequence libraries. Though many aptamers for a broad range of targets with high affinity and selectivity have been generated, a lack of high-resolution structural data and the limitations of currently available biophysical tools greatly impede understanding of the mechanisms of aptamer-ligand interactions. Here we demonstrate that an approach based on native electrospray ionization mass spectrometry (ESI-MS) can be successfully applied to characterize aptamer-ligand complexes in all details. We studied an adenosine-binding aptamer (ABA), a l-argininamide-binding aptamer (LABA), and a cocaine-binding aptamer (CBA) and their noncovalent interactions with ligands by native ESI-MS and complemented these measurements by ion mobility spectrometry (IMS), isothermal titration calorimetry (ITC), and circular dichroism (CD) spectroscopy. The ligand selectivity of the aptamers and the respective complex stoichiometry could be determined by the native ESI-MS approach. The ESI-MS data can also help refining the binding model for aptamer-ligand complexes and deliver accurate aptamer-ligand binding affinities for specific and nonspecific binding events. For specific ligands, we found Kd1 = 69.7 μM and Kd2 = 5.3 μM for ABA (two binding sites); Kd1 = 22.04 μM for LABA; and Kd1 = 8.5 μM for CBA.