In modern biochemistry, protein stability and ligand interactions are of high interest. These properties are often studied with methods requiring labeled biomolecules, as the existing methods utilizing luminescent external probes suffer from low sensitivity. Currently available label-free technologies, e.g., thermal shift assays, circular dichroism, and differential scanning calorimetry, enable studies on protein unfolding and protein–ligand interactions (PLI). Unfortunately, the required micromolar protein concentration increases the costs and predisposes these methods for spontaneous protein aggregation. Here, we report a time-resolved luminescence method for protein unfolding and PLI detection with nanomolar sensitivity. The Protein-Probe method is based on highly luminescent europium chelate-conjugated probe, which is the key component in sensing the hydrophobic regions exposed to solution after protein unfolding. With the same Eu-probe, we also demonstrate ligand-interaction induced thermal stabilization with model proteins. The developed Protein-Probe method provides a sensitive approach overcoming the problems of the current label-free methodologies.

Protein–protein interactions (PPIs) are an essential part of correct cellular functionality, making them increasingly interesting drug targets. While Förster resonance energy transfer-based methods have traditionally been widely used for PPI studies, label-free techniques have recently drawn significant attention. These methods are ideal for studying PPIs, most importantly as there is no need for labeling of either interaction partner, reducing potential interferences and overall costs. Already, several different label-free methods are available, such as differential scanning calorimetry and surface plasmon resonance, but these biophysical methods suffer from low to medium throughput, which reduces suitability for high-throughput screening (HTS) of PPI inhibitors. Differential scanning fluorimetry, utilizing external fluorescent probes, is an HTS compatible technique, but high protein concentration is needed for experiments. To improve the current concepts, we have developed a method based on time-resolved luminescence, enabling PPI monitoring even at low nanomolar protein concentrations. This method, called the protein probe technique, is based on a peptide conjugated with Eu 3+ chelate, and it has already been applied to monitor protein structural changes and small molecule interactions at elevated temperatures. Here, the applicability of the protein probe technique was demonstrated by monitoring single-protein pairing and multiprotein complexes at room and elevated temperatures. The concept functionality was proven by using both artificial and multiple natural protein pairs, such as KRAS and eIF4A together with their binding partners, and C-reactive protein in a complex with its antibody.

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein’s physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a ‘Gold Standard’ for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins.

Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and localization of many proteins, and their dysregulation is associated with various pathological conditions. Proteases have been identified as biomarkers and potential therapeutic targets for multiple diseases, such as acquired immunodeficiency syndrome, cardiovascular diseases, osteoporosis, type 2 diabetes, and cancer, where they are essential to disease progression. Thus, protease inhibitors and inhibitor-like molecules are interesting drug candidates. To study proteases and their substrates and inhibitors, simple, rapid, and sensitive protease activity assays are needed. Existing fluorescence-based assays enable protease monitoring in a high-throughput compatible microtiter plate format, but the methods often rely on either molecular labeling or synthetic protease targets that only mimic the hydrolysis site of the true target proteins. Here, we present a homogenous, label-free, and time-resolved luminescence utilizing the protein-probe method to assay proteases with native and denatured substrates at nanomolar sensitivity. The developed protein-probe method is not restricted to any single protein or protein target class, enabling digestion and substrate fragmentation studies with the natural unmodified substrate proteins. The versatility of the assay for studying protease targets was shown by monitoring the digestion of a substrate panel with different proteases. These results indicate that the protein-probe method not only monitors the protease activity and inhibition, but also studies the substrate specificity of individual proteases.

Protein aggregation is a spontaneous process affected by multiple external and internal properties, such as buffer composition and storage temperature. Aggregation of protein-based drugs can endanger patient safety due, for example, to increased immunogenicity. Aggregation can also inactivate protein drugs and prevent target engagement, and thus regulatory requirements are strict regarding drug stability monitoring during manufacturing and storage. Many of the current technologies for aggregation monitoring are time- and material-consuming and require specific instruments and expertise. These types of assays are not only expensive, but also unsuitable for larger sample panels. Here we report a label-free time-resolved luminescence-based method using an external Eu 3+ -conjugated probe for the simple and fast detection of protein stability and aggregation. We focused on monitoring the properties of IgG, which is a common format for biological drugs. The Protein-Probe assay enables IgG aggregation detection with a simple single-well mix-and-measure assay performed at room temperature. Further information can be obtained in a thermal ramping, where IgG thermal stability is monitored. We showed that with the Protein-Probe, trastuzumab aggregation was detected already after 18 hours of storage at 60°C, 4 to 8 days earlier compared to SYPRO Orange- and UV250-based assays, respectively. The ultra-high sensitivity of less than 0.1% IgG aggregates enables the Protein-Probe to reduce assay time and material consumption compared to existing techniques.

Post-translational modifications (PTMs) of proteins provide an important mechanism for cell signal transduction control. Impaired PTM control is a key feature in multiple different disease states, and thus the enzyme-controlling PTMs have drawn attention as highly promising drug targets. Due to the importance of PTMs, various methods to monitor PTM enzyme activity have been developed, but universal high-throughput screening (HTS), a compatible method for different PTMs, remains elusive. Here, we present a homogeneous single-label thermal dissociation assay for the detection of enzymatic PTM removal. The developed method allows the use of micromolar concentration of substrate peptide, which is expected to be beneficial when monitoring enzymes with low activity and peptide binding affinity. We prove the thermal dissociation concept functionality using peptides for dephosphorylation, deacetylation, and demethylation and demonstrate the HTS-compatible flash isothermal method for PTM enzyme activity monitoring. Using specific inhibitors, we detected literature-comparable IC50 values and Z′ factors from 0.61 to 0.72, proving the HTS compatibility of the thermal peptide-break technology.

This diagnostic study assesses the use of dried blood samples to detect prohormone brain-type natriuretic peptide for diagnosis of congenital heart disease in neonates in Sweden.

Viruses play a major role in modern society and create risks from global pandemics and bioterrorism to challenges in agriculture. Virus infectivity assays and genome copy number determination methods are often used to obtain information on virus preparations used in diagnostics and vaccine development. However, these methods do not provide information on virus particle count. Current methods to measure the number of viral particles are often cumbersome and require highly purified virus preparations and expensive instrumentation. To tackle these problems, we developed a simple and cost-effective time-resolved luminescence-based method for virus particle quantification. This mix-and-measure technique is based on the recognition of the virus particles by an external Eu3+-peptide probe, providing results on virus count in minutes. The method enables the detection of non-enveloped and enveloped viruses, having over tenfold higher detectability for enveloped, dynamic range from 5E6 to 3E10 vp/mL, than non-enveloped viruses. Multiple non-enveloped and enveloped viruses were used to demonstrate the functionality and robustness of the Protein-Probe method. Graphical abstract