Homogeneous peptide-break assay for luminescent detection of enzymatic protein post-translational modification activity utilizing charged peptides

Kopra, Kari; Tong-Ochoa, Natalia; Laine, Mari; Eskonen, Ville; Koskinen, Päivi J.; Härmä, Harri

doi:10.1016/j.aca.2018.12.041

Cited by 4 publications

(19 citation statements)

References 38 publications

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“…Consequently, modification of the substrate peptide disintegrates this interaction reducing the monitored TRL-signal. 15 , 17 − 19 These methods based on the peptide pairing via coiled-coil leucine-zipper or charged amino acid residues were suitable for different types of PTMs, but there are limitations in using natural substrates. Therefore, we aimed to develop a concept where structural and sequence constrains are less pronounced and the substrate sequence from the natural source of the PTM can be more freely selected.…”

Section: Introductionmentioning

confidence: 99%

“…Using lanthanide donor, higher sensitivity, and robustness in biological matrixes is obtained, and thus multiple commercial assays for several analytes utilize the method . Previously, we have introduced sensitive antibody-free methods for the detection of several PTMs utilizing the TRL-signal readout. − These methods were based on two interacting peptides leading to a TRL-signal shielded from soluble quencher molecules as a result of the paired peptide structure. Consequently, modification of the substrate peptide disintegrates this interaction reducing the monitored TRL-signal. ,− These methods based on the peptide pairing via coiled-coil leucine-zipper or charged amino acid residues were suitable for different types of PTMs, but there are limitations in using natural substrates.…”

Section: Introductionmentioning

confidence: 99%

“…Previously, we have introduced sensitive antibody-free methods for the detection of several PTMs utilizing the TRL-signal readout. − These methods were based on two interacting peptides leading to a TRL-signal shielded from soluble quencher molecules as a result of the paired peptide structure. Consequently, modification of the substrate peptide disintegrates this interaction reducing the monitored TRL-signal. ,− These methods based on the peptide pairing via coiled-coil leucine-zipper or charged amino acid residues were suitable for different types of PTMs, but there are limitations in using natural substrates. Therefore, we aimed to develop a concept where structural and sequence constrains are less pronounced and the substrate sequence from the natural source of the PTM can be more freely selected.…”

Section: Introductionmentioning

confidence: 99%

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Single-Peptide TR-FRET Detection Platform for Cysteine-Specific Post-Translational Modifications

et al. 2020

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Post-translational modifications (PTMs) are one of the most important regulatory mechanisms in cells, and they play key roles in cell signaling both in health and disease. PTM catalyzing enzymes have become significant drug targets, and therefore, tremendous interest has been focused on the development of broad-scale assays to monitor several different PTMs with a single detection platform. Most of the current methodologies suffer from low throughput or rely on antibody recognition, increasing the assay costs, and decreasing the multifunctionality of the assay. Thus, we have developed a sensitive time-resolved Förster resonance energy transfer (TR-FRET) detection method for PTMs of cysteine residues using a single-peptide approach performed in a 384-well format. In the developed assay, the enzyme-specific biotinylated substrate peptide is post-translationally modified at the cysteine residue, preventing the subsequent thiol coupling with a reactive AlexaFluor 680 acceptor dye. In the absence of enzymatic activity, increase in the TR-FRET signal between the biotin-bound Eu(III)-labeled streptavidin donor and the cysteine-coupled AlexaFluor 680 acceptor dye is observed. We demonstrate the detection concept with cysteine modifying S-nitrosylation and ADP-ribosylation reactions using a chemical nitric oxide donor S-nitrosoglutathione and enzymatic ADP-ribosyltransferase PtxS1-subunit of pertussis toxin, respectively. As a proof of concept, three peptide substrates derived from the small GTPase K-Ras and the inhibitory α-subunit of the heterotrimeric G-protein Gαi showed expected functionality in both chemical and enzymatic assays. Measurements yielded signal-to-background ratios of 28.7, 33.0, and 8.7 between the modified and the nonmodified substrates for the three peptides in the S-nitrosylation assay, 5.8 in the NAD + hydrolysis assay, and 6.8 in the enzymatic ADP-ribosyltransferase inhibitor dose–response assay. The developed antibody-free assay for cysteine-modifying enzymes provides a detection platform with low nanomolar peptide substrate consumption, and the assay is potentially applicable to investigate various cysteine-modifying enzymes in a high throughput compatible format.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Single-Peptide TR-FRET Detection Platform for Cysteine-Specific Post-Translational Modifications

et al. 2020

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“…Previously, we have developed an antibody-free PTM monitoring method called the peptide-break technology which allows the detection of a variety of PTMs in a single platform. − The peptide-break technology is based on the peptide dimerization monitored using the single-label quenching resonance energy transfer (QRET) technology demonstrated previously for the detection of various PTM targets. , Peptide dimer is formed between the Eu 3+ -labeled detection peptide and the selected substrate peptide designed for the studied PTM enzyme. In the absence of PTM, peptides form a complex leading to high TRL signal due to Eu 3+ -chelate protection from the soluble quenchers, while the PTM addition to the substrate peptide dissociates the peptide complex and the TRL signal is quenched.…”

Section: Introductionmentioning

confidence: 99%

“…In the absence of PTM, peptides form a complex leading to high TRL signal due to Eu 3+ -chelate protection from the soluble quenchers, while the PTM addition to the substrate peptide dissociates the peptide complex and the TRL signal is quenched. We have demonstrated that the peptide complex can be formed using either specific leucine zippers or merely charge-based interactions. − This concept provides versatility to the system and allows assay optimizations of a variety of PTM types using nanomolar concentration of reagents.…”

Section: Introductionmentioning

confidence: 99%

Thermal Dissociation Assay for Time-Resolved Fluorescence Detection of Protein Post-Translational Modifications

et al. 2019

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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.

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Label-free electronic detection of peptide post-translational modification with functional enzyme-driven assay at the physical limit

Macchia,

Björkström,

Tewari

et al. 2024

Cell Reports Physical Science

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Homogeneous peptide-break assay for luminescent detection of enzymatic protein post-translational modification activity utilizing charged peptides

Cited by 4 publications

References 38 publications

Single-Peptide TR-FRET Detection Platform for Cysteine-Specific Post-Translational Modifications

Single-Peptide TR-FRET Detection Platform for Cysteine-Specific Post-Translational Modifications

Thermal Dissociation Assay for Time-Resolved Fluorescence Detection of Protein Post-Translational Modifications

Label-free electronic detection of peptide post-translational modification with functional enzyme-driven assay at the physical limit

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