Optimization of peptide substrates for botulinum neurotoxin E improves detection sensitivity in the Endopep–MS assay

Wang, Dongxia; Krilich, Joan; Baudys, Jakub; Barr, John R.; Kalb, Suzanne R.

doi:10.1016/j.ab.2014.08.026

Cited by 11 publications

(9 citation statements)

References 25 publications

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“…Inserts show the isotope peak pattern of cleaved peptides if detectable. The occurrence of only the C -terminal peptide fragment in the BoNT/E control sample is due to an instability of this peptide substrate, which was already previously recognized [ 40 , 41 ]. To circumvent this signal, very recently, an optimized peptide substrate for BoNT/E was introduced by incorporating a non-natural homo-arginine residue at P1 position (first AA position upstream of the cleavage site), as well as introducing C -terminal amidation of the peptide substrate, which significantly reduced unspecific cleavage and increased peptide stability [ 41 ].…”

Section: Resultsmentioning

confidence: 87%

Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test

Weisemann¹,

Krez²,

Fiebig

et al. 2015

Toxins

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The detection and identification of botulinum neurotoxins (BoNT) is complex due to the existence of seven serotypes, derived mosaic toxins and more than 40 subtypes. Expert laboratories currently use different technical approaches to detect, identify and quantify BoNT, but due to the lack of (certified) reference materials, analytical results can hardly be compared. In this study, the six BoNT/A1–F1 prototypes were successfully produced by recombinant techniques, facilitating handling, as well as improving purity, yield, reproducibility and biosafety. All six BoNTs were quantitatively nicked into active di-chain toxins linked by a disulfide bridge. The materials were thoroughly characterized with respect to purity, identity, protein concentration, catalytic and biological activities. For BoNT/A1, B1 and E1, serotypes pathogenic to humans, the catalytic activity and the precise protein concentration were determined by Endopep-mass spectrometry and validated amino acid analysis, respectively. In addition, BoNT/A1, B1, E1 and F1 were successfully detected by immunological assays, unambiguously identified by mass spectrometric-based methods, and their specific activities were assigned by the mouse LD50 bioassay. The potencies of all six BoNT/A1–F1 were quantified by the ex vivo mouse phrenic nerve hemidiaphragm assay, allowing a direct comparison. In conclusion, highly pure recombinant BoNT reference materials were produced, thoroughly characterized and employed as spiking material in a worldwide BoNT proficiency test organized by the EQuATox consortium.

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Section: Resultsmentioning

confidence: 87%

Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test

Weisemann¹,

Krez²,

Fiebig

et al. 2015

Toxins

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“…BoNT/E-Lc has the unique property of cleaving the specific peptide bonds of SNAP-25 because BoNT/E-Lc is a zinc-metalloprotease that hydrolyzes the bond between arginine (R) 180 and isoleucine (I) 181 in the SNAP-25 sequence [21,22,35]. Using these properties, we attempted to detect BoNT/E-Lc on the previously prepared all-CNT-based FET devices.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, field-effect transistor (FET)-based bioelectronics, involving the transduction of signals from the biological system to electrical signals at the bio-electronics interface, has been intensively investigated in various areas [19,20,21,22,23,24]. Owing to ultrasensitive detection and high-throughput, such real-time embedded systems potentially improve the fundamental understanding of biological phenomena and allow development of biomedical devices such as cardiac pacemakers, deep-brain stimulators, and blood glucose sensors.…”

Section: Introductionmentioning

confidence: 99%

Real-Time Monitoring of a Botulinum Neurotoxin Using All-Carbon Nanotube-Based Field-Effect Transistor Devices

Lee

Nahm

Choi

2018

Sensors

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The possibility of exposure to botulinum neurotoxin (BoNT), a powerful and potential bioterrorism agent, is considered to be ever increasing. The current gold-standard assay, live-mouse lethality, exhibits high sensitivity but has limitations including long assay times, whereas other assays evince rapidity but lack factors such as real-time monitoring or portability. In this study, we aimed to devise a novel detection system that could detect BoNT at below-nanomolar concentrations in the form of a stretchable biosensor. We used a field-effect transistor with a p-type channel and electrodes, along with a channel comprising aligned carbon nanotube layers to detect the type E light chain of BoNT (BoNT/E-Lc). The detection of BoNT/E-Lc entailed observing the cleavage of a unique peptide and the specific bonding between BoNT/E-Lc and antibody BoNT/E-Lc (Anti-BoNT/E-Lc). The unique peptide was cleaved by 60 pM BoNT/E-Lc; notably, 52 fM BoNT/E-Lc was detected within 1 min in the device with the antibody in the bent state. These results demonstrated that an all-carbon nanotube-based device (all-CNT-based device) could be produced without a complicated fabrication process and could be used as a biosensor with high sensitivity, suggesting its potential development as a wearable BoNT biosensor.

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“…However, these tests are less convenient than the direct and automated detection method presented in this paper. Indeed, they are based on a two-step approach involving toxin concentration/purification prior to assaying toxin activity 8 16 25 26 .…”

Section: Discussionmentioning

confidence: 99%

An optical biosensor assay for rapid dual detection of Botulinum neurotoxins A and E

Lévêque

Ferracci

Maulet

et al. 2015

Sci Rep

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The enzymatic activity of the pathogenic botulinum neurotoxins type A and E (BoNT/A and E) leads to potentially lethal paralytic symptoms in humans and their prompt detection is of crucial importance. A chip assay based on Surface Plasmon Resonance monitoring of the cleavage products is a simple method that we have previously established to detect BoNT/A activity. We have now developed a similar format assay to measure BoNT/E activity. A monoclonal antibody specifically recognizing SNAP25 cleaved by BoNT/E was generated and used to measure the appearance of the neo-epitope following injection of BoNT/E over SNAP-25 immobilized on a chip. This assay detects BoNT/E activity at 1 LD50/ml within minutes and linear dose-responses curves were obtained using a multiplexed biosensor. A threshold of 0.01 LD50/ml was achieved after 5 h of cleavage. This assay is 10-fold more sensitive than the in vivo assay for direct detection of BoNT/E in serum samples. The SNAP25 chip assay is able to discriminate in an automated manner the presence of BoNT/E, BoNT/A or a combination of both toxins.

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Optimization of peptide substrates for botulinum neurotoxin E improves detection sensitivity in the Endopep–MS assay

Cited by 11 publications

References 25 publications

Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test

Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test

Real-Time Monitoring of a Botulinum Neurotoxin Using All-Carbon Nanotube-Based Field-Effect Transistor Devices

An optical biosensor assay for rapid dual detection of Botulinum neurotoxins A and E

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