Sensitive Label-Free Thermal Stability Assay for Protein Denaturation and Protein–Ligand Interaction Studies

Vuorinen, Emmiliisa; Valtonen, Salla; Eskonen, Ville; Kariniemi, Taru; Jakovleva, Jelena; Kopra, Kari; Härmä, Harri

doi:10.1021/acs.analchem.9b05712

Cited by 20 publications

(37 citation statements)

References 28 publications

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“…Interactions between proteins are a fundamental part of correct cellular functionality, and impaired interactions may lead to various disease states. We have previously developed a label-free method for the detection of protein stability and PLIs, 27 which we now apply for PPI monitoring. The protein probe technique is based on a negatively charged Eu 3+ chelate-labeled peptide (Eu 3+ probe) used to measure protein structural changes, such as an increased surface area and exposed hydrophobic regions.…”

Section: Resultsmentioning

confidence: 99%

“…We have previously shown that an increase in the TRL signal is observed when a heated and denatured protein sample is monitored with the protein probe method, and we expected to measure an improved detectability with PPIs. 27 In all assays, the reactions are performed in biologically relevant buffers at RT, and thereafter the protein complex is heated to a given temperature. For the detection, the protein solution is reverted to RT by adding the protein probe solution.…”

Section: Resultsmentioning

confidence: 99%

“… 26 In addition, we have recently introduced an external Eu 3+ -labeled protein probe technique for the detection of protein stability and PLIs at a low nanomolar sensitivity level. 27 In this study, we introduce a further development of the protein probe for the detection of PPIs. The method is applicable not only for thermal but also for isothermal PPI detection, depending on the nature of the studied proteins or protein complexes.…”

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confidence: 99%

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Nanomolar Protein–Protein Interaction Monitoring with a Label-Free Protein-Probe Technique

et al. 2020

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Nanomolar Protein–Protein Interaction Monitoring with a Label-Free Protein-Probe Technique

et al. 2020

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“…The Eu-probe was purified as described before, [31] and the concentration was determined using the DELFIA technique and a commercial EuCl 3 standard from PerkinElmer Life and Analytical Sciences, Wallac (Turku, Finland). The protein substrates, recombinant pertussis toxin (PTX), G protein alpha I subunit (Gαi) [33], human Ras GTPase-activating protein 1 (p120GAP) [34], wild type Kirsten RAt Sarcoma virus (KRAS, 2-188), human son of sevenless SOS1 catalytic domain (SOS cat , 564-1048) [35], and eukaryotic initiation factor 4A1 (eIF4A1) [32] were kind gifts from our collaborators. Malate dehydrogenase (MDH) was purchased from Roche (Basel, Switzerland).…”

Section: Materials Instrumentation and Assay Buffersmentioning

confidence: 99%

Protease Substrate-Independent Universal Assay for Monitoring Digestion of Native Unmodified Proteins

Vuorinen

Valtonen

Hassan

et al. 2021

IJMS

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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.

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“…In addition, our Protein-Probe method requires only a fraction of the sample compared to other introduced methods, and it is also suitable for protein stability monitoring, giving additional information when assayed with thermal ramping. 35 , 36 Here, we used the Protein-Probe in a proof-of-principle study to monitor a panel of monoclonal antibodies (mAbs) and their heat- or pH-induced aggregation (RT measurements), and also stability in thermal ramping. Both monitored parameters – aggregation behavior and thermal stability – can give important information on therapeutic mAbs.…”

Section: Introductionmentioning

confidence: 99%

Sensitive, homogeneous, and label-free protein-probe assay for antibody aggregation and thermal stability studies

Valtonen

Vuorinen

Eskonen

et al. 2021

mAbs

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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.

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Sensitive Label-Free Thermal Stability Assay for Protein Denaturation and Protein–Ligand Interaction Studies

Cited by 20 publications

References 28 publications

Nanomolar Protein–Protein Interaction Monitoring with a Label-Free Protein-Probe Technique

Nanomolar Protein–Protein Interaction Monitoring with a Label-Free Protein-Probe Technique

Protease Substrate-Independent Universal Assay for Monitoring Digestion of Native Unmodified Proteins

Sensitive, homogeneous, and label-free protein-probe assay for antibody aggregation and thermal stability studies

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