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

Valtonen, Salla; Vuorinen, Emmiliisa; Eskonen, Ville; Malakoutikhah, Morteza; Kopra, Kari; Härmä, Harri

doi:10.1080/19420862.2021.1955810

Cited by 8 publications

(8 citation statements)

References 44 publications

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“…We previously developed a novel DSF-like method called the Protein-Probe [19][20][21][22]. The method is based on a Eu 3+ -labeled peptide probe, where a negatively charged peptide has minimal interaction with a folded protein, and the time-resolved luminescence (TRL) signal is low in the modulation solution containing a cyanine dye as quencher.…”

Section: Introductionmentioning

confidence: 99%

“…This technique can avoid many of the problems related to conventional DSF dyes. The Protein-Probe enables similar stability, interaction, and buffer solution composition studies as conventional external thermal dyes, but with significantly improved sensitivity [19][20][21][22]. In addition, we have extensively studied KRAS and other GTPase proteins to enable monitoring of their functional properties [23][24][25][26].…”

Section: Introductionmentioning

confidence: 99%

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Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability

Kopra

Valtonen

Mahran

et al. 2022

IJMS

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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.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability

Kopra

Valtonen

Mahran

et al. 2022

IJMS

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“…Unfortunately, most of the used methods are time-consuming and expensive, as well as potentially require special instrumentation and expertise. We have previously introduced a label-free technique called the Protein-Probe, which has been applied for varying protein-related studies [ 35 – 38 ]. Using the protein-binding property of the Eu 3+ -probe, a key component of the Protein-Probe technique, we have now developed a virus particle quantitation assay, exploiting viral surface proteins.…”

Section: Resultsmentioning

confidence: 99%

“…In this paper, we present a novel virus particle counting method utilizing the recently developed label-free Protein-Probe technique [ 35 – 38 ]. In the Protein-Probe technique, the external Eu 3+ -peptide probe senses the concentration of monitored virus particles by an increase in the time-resolved luminescence (TRL) signal.…”

Section: Introductionmentioning

confidence: 99%

Rapid high-throughput compatible label-free virus particle quantification method based on time-resolved luminescence

et al. 2022

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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

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“…As with other DSF-type methods, thermal denaturation exposes protein inner regions and promotes the binding of the Eu-probe, which can be monitored with an increase in TRL-signal (Figure ). We have demonstrated that the protein-probe method is highly sensitive and can detect protein interactions, stability, aggregation, and enzyme activities with 100-fold higher sensitivity than the commonly used SYPRO Orange. ,− In this study, we describe our scientific efforts to further improve the sensitivity and simplicity of the assay by modifying the quencher structure; 13 quencher molecules were prepared and studied using the protein-probe platform. Our results demonstrate significant improvements in assay performance, enabling the detection of protein stability and protein–ligand interaction using a one-step peptide-probe protocol at neutral pH buffers (Figure ).…”

Section: Introductionmentioning

confidence: 99%

Nanomolar Protein Thermal Profiling with Modified Cyanine Dyes

Malakoutikhah,

Mahran,

Gooran

et al. 2023

Anal. Chem.

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Protein properties and interactions have been widely investigated by using external labels. However, the micromolar sensitivity of the current dyes limits their applicability due to the high material consumption and assay cost. In response to this challenge, we synthesized a series of cyanine5 (Cy5) dye-based quencher molecules to develop an external dye technique to probe proteins at the nanomolar protein level in a high-throughput one-step assay format. Several families of Cy5 dye-based quenchers with ring and/or side-chain modifications were designed and synthesized by introducing organic small molecules or peptides. Our results showed that steric hindrance and electrostatic interactions are more important than hydrophobicity in the interaction between the luminescent negatively charged europium-chelate-labeled peptide (Eu-probe) and the quencher molecules. The presence of substituents on the quencher indolenine rings reduces their quenching property, whereas the increased positive charge on the indolenine side chain improved the interaction between the quenchers and the luminescent compound. The designed quencher structures entirely altered the dynamics of the Eu-probe (protein-probe) for studying protein stability and interactions, as we were able to reduce the quencher concentration 100-fold. Moreover, the new quencher molecules allowed us to conduct the experiments using neutral buffer conditions, known as the peptide-probe assay. These improvements enabled us to apply the method in a one-step format for nanomolar protein–ligand interaction and protein profiling studies instead of the previously developed two-step protocol. These improvements provide a faster and simpler method with lower material consumption.

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

Cited by 8 publications

References 44 publications

Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability

Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability

Rapid high-throughput compatible label-free virus particle quantification method based on time-resolved luminescence

Nanomolar Protein Thermal Profiling with Modified Cyanine Dyes

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