Improved detection of botulinum neurotoxin serotype A by Endopep–MS through peptide substrate modification

Botulinum neurotoxins (BoNTs) are a family of seven toxin serotypes that are the most toxic substances known to humans. Intoxication with BoNT causes flaccid paralysis and can lead to death if untreated with serotype-specific antibodies. Supportive care, including ventilation, may be necessary. Rapid and sensitive detection of BoNT is necessary for timely clinical confirmation of clinical botulism. Previously, our laboratory developed a fast and sensitive mass spectrometry (MS) method termed the Endopep–MS assay. The BoNT serotypes are rapidly detected and differentiated by extracting the toxin with serotype-specific antibodies and detecting the unique and serotype-specific cleavage products of peptide substrates that mimic the sequence of the BoNT native targets. To further improve the sensitivity of the Endopep–MS assay, we report here the optimization of the substrate peptide for the detection of BoNT/A. Modifications on the terminal groups of the original peptide substrate with acetylation and amidation significantly improved the detection of BoNT/A cleavage products. The replacement of some internal amino acid residues with single or multiple substitutions led to further improvement. An optimized peptide increased assay sensitivity 5-fold with toxin spiked into buffer solution or different biological matrices.     

Improved detection of botulinum type E by rational design of a new peptide substrate for endopeptidase–mass spectrometry assay

Botulinum neurotoxins (BoNTs) are the most toxic substances known to humans. Endopeptidase–mass spectrometry (Endopep–MS) is used as a specific and rapid in vitro assay to detect BoNTs. In this assay, immunocaptured toxin cleaves a serotype-specific peptide substrate, and the cleavage products are then detected by MS. To further improve the sensitivity of the assay, we report here the rational design of a new substrate peptide for the detection of botulinum neurotoxin type E (BoNT/E). Our strategy was based on previously reported structural interactions integrated with analysis method efficiency considerations. Integration of the newly designed substrate has led to a more than one order of magnitude increased sensitivity of the assay.     

A new peptide substrate for enhanced botulinum neurotoxin type B detection by endopeptidase–liquid chromatography–tandem mass spectrometry/multiple reaction monitoring assay

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Rapid and sensitive detection of BoNTs is achieved by the endopeptidase–mass spectrometry (Endopep–MS) assay. In this assay, BoNT cleaves a specific peptide substrate and the cleaved products are analyzed by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type B (BoNT/B) in the Endopep–MS assay. Our strategy was based on reported BoNT/B–substrate interactions integrated with analysis method efficiency considerations. Incorporation of the new peptide led to a 5-fold increased sensitivity of the assay both in buffer and in a clinically relevant human spiked serum.     

Characterization of Förster resonance energy transfer in a botulinum neurotoxin protease assay

Using the cell-permeable, radioiodinated, irreversible inhibitor BIL-DMK, we probed active cysteine cathepsins in blood. Incubation of the probe in human whole blood followed by separation of white blood cells by dextran sedimentation led to the labeling of one major band at 24 kDa. Two-dimensional gel electrophoresis showed that the band resolved in a single protein spot and corresponded to cathepsin S based on its molecular mass, isoelectric point, and Western blot analysis using anti-human cathepsin S antibodies. Cathepsin S activity in human whole blood was dependent on the time of blood collection, suggesting that cathepsin S activity is subject to circadian variations. Separation of white blood cell populations using a magnetic cell sorter and further characterization by FACS (fluorescent-activated cell sorting) analysis demonstrated that the majority of active cathepsin S resided in the monocyte and neutrophil populations, whereas on a cell basis cathepsin S activity in granulocytes is 10-fold lower than that in monocytes. A whole blood cathepsin S assay was developed and used to measure cathepsin S inhibition in both in vitro and ex vivo conditions. To determine the correlation between the in vitro and ex vivo assays, a reversible cathepsin S inhibitor was dosed intravenously to a rhesus monkey. The inhibitor concentration required to inhibit 50% of the cathepsin S activity ex vivo correlated well with the concentration required to inhibit the enzyme in rhesus monkey whole blood in vitro. The results reported here demonstrate the utility of the activity-based probe BIL-DMK for the ex vivo assessment of cathepsin S inhibition.     

Investigation of an on-line two-dimensional chromatographic approach for peptide analysis in plasma by LC–MS–MS

Reversed phase and hydrophilic interaction chromatography (HILIC) were successfully coupled for the on-line extraction and quantitative analysis of peptides by ESI–LC–MS/MS. A total of 11 peptides were utilized to determine the conditions for proper focusing and separation on both dimensions. Minor modifications to the initial organic composition of the first reversed-phase dimension provided options between a comprehensive (generic) or more selective approach for peptide transfer to the second HILIC dimension. Ion-pairing with trifluoroacetic acid (TFA) provided adequate chromatographic retention and peak symmetry for the selected peptides on both C₁₈ and HILIC. The resulting signal suppression from TFA was partially recovered by a post-column “TFA fix” using acetic acid yielding improvements in sensitivity. Minimal sample preparation aligned with standard on-line extraction procedures provided highly reproducible and robust results for over 300 sequential matrix injections. Final optimized conditions were successfully employed for the quantitation of peptide PTHrP (1–36) in rat K₃EDTA plasma from 25.0 to 10,000 ng/mL using PTHrP (1–34) as the analog internal standard. This highly orthogonal two-dimensional configuration was found to provide the unique selectivity required to overcome issues with interfering endogenous components and minimize electrospray ionization effects in biological samples.     

Rapid and sensitive LC–MS/MS assay for the quantitation of 20(S)-protopanaxadiol in human plasma

This paper describes a rapid and sensitive method for the quantitation of 20(S)-protopanaxadiol (PPD) in human plasma based on high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and internal standard (I.S.), ginsenoside Rh₂, were extracted from plasma by liquid–liquid extraction and separated on a Zorbax extend C₁₈ analytical column using methanol–acetonitrile-10 mM ammonium acetate (47.5:47.5:5, v/v/v) as mobile phase. Detection was by tandem mass spectrometry using electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM). The assay was linear over the concentration range 0.1–100.0 ng/ml with a limit of detection of 0.05 ng/ml. The method was successfully applied to a clinical pharmacokinetic study in healthy volunteers after a single oral administration of a PPD 25 mg capsule.     

A multi-component LC–MS/MS method for detection of ten plant-derived psychoactive substances in urine

A sensitive and specific LC–MS/MS method for simultaneous detection of 10 plant-derived psychoactive substances (atropine, N,N-dimethyltryptamine, ephedrine, harmaline, harmine, ibogaine, lysergic acid amide, psilocin, scopolamine and yohimbine) in urine was developed. Direct injection of urine diluted with 3 deuterated internal standards allowed for a readily accessible method suitable for application in clinical intoxication cases. Separation was achieved using reversed phase chromatography and gradient elution with a total analysis time of 14 min. Electrospray ionization was used and ions were monitored in the positive selected reaction monitoring mode. The calibration curves were linear (r² > 0.999) and the total imprecision at high (1000 μg/L) and low (50 μg/L) substance concentrations were 4.9–13.8% and 8.3–26%, respectively. Infusing the analytes post column and injecting matrix samples showed limited influence by ion suppression. The multi-component method proved to be useful for investigation of authentic cases of intoxication with plant-derived psychoactive drugs and was indicated to cover the clinically relevant concentration ranges.     

Non-destructive assay systems for detection of β-glucuronidase activity in higher plants

β-glucuronidase (GUS) can be qualitatively assayed in seedlings and fully grown plants without injury or irreversible damage by short term incubations in X-gluc or by spraying 4-MUG.     

Small molecule non-peptide inhibitors of botulinum neurotoxin serotype E: Structure–activity relationship and a pharmacophore model

Botulinum neurotoxins (BoNTs) are the most poisonous biological substance known to humans. They cause flaccid paralysis by blocking the release of acetylcholine at the neuromuscular junction. Here, we report a number of small molecule non-peptide inhibitors of BoNT serotype E. The structure–activity relationship and a pharmacophore model are presented. Although non-peptidic in nature, these inhibitors mimic key features of the uncleavable substrate peptide Arg-Ile-Met-Glu (RIME) of the SNAP-25 protein. Among the compounds tested, most of the potent inhibitors bear a zinc-chelating moiety connected to a hydrophobic and aromatic moiety through a carboxyl or amide linker. All of them show low micromolar IC₅₀ values.     

Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase–peroxygenase

Agrocybe aegerita peroxidase (AaP) is a versatile heme-thiolate protein that can act as a peroxygenase and catalyzes, among other reactions, the hydroxylation of aromatic rings. This paper reports a rapid and selective spectrophotometric method for directly detecting aromatic hydroxylation by AaP. The weakly activated aromatic compound naphthalene served as the substrate that was regioselectively converted into 1-naphthol in the presence of the co-substrate hydrogen peroxide. Formation of 1-naphthol was followed at 303 nm (ɛ ₃₀₃ = 2,010 M⁻¹ cm⁻¹), and the apparent Michaelis–Menten (K _m) and catalytic (k _cat) constants for the reaction were estimated to be 320 μM and 166 s⁻¹, respectively. This method will be useful in screening of fungi and other microorganisms for extracellular peroxygenase activities and in comparing and assessing different catalytic activities of haloperoxidase–peroxygenases.     

Optimization of extraction method for LC–MS based determination of phenolic acid profiles in different Impatiens species

The main aim of presented study was the comparison of various extraction methods for the quantitative and qualitative analysis (LC-ESI–MS/MS) of phenolic acids present in extracts obtained from leaves, flowers, and roots of Impatiens glandulifera. The accelerated solvent extraction (ASE) at three temperature ranges (80° C, 100° C, and 120° C), ultrasound assisted extraction (USAE) at 60° C, and traditional extraction in Soxhlet apparatus were used. Taking into account the extraction yield, and the diversity of the individual compounds, ultrasound assisted extraction proved to be the most efficient method, and it was used to determine the content of phenolic acids in leaves of four other Impatiens species, including I. balsamina, I. noli-tangere, I. parviflora, and I. walleriana. Eleven phenolic acids were identified in all examined species. These were protocatechuic, gentisic, 4- hydroxybenzoic, vanillic, trans-caffeic, syringic, trans-p-coumaric, trans- and cis-ferulic, salicylic, and 3-hydroxycinnamic acids. In the extract from the leaves of I. balsamina and I. walleriana, gallic and cis-p-coumaric acids were found additionally. The most abundant compounds in all examined extracts were protocatechuic and 3-hydroxycinnamic acids. The latest acid was found in the highest yield in I. noli-tangere (266.12 μg/g DW). In the leaves of I. glandulifera a great amount of 4-hydroxybenzoic (41.44 μg/g DW), vanillic (61.50 μg/g DW), and trans-p-coumaric (58.42 μg/g DW) acids was also observed. Our results indicate that protocatechuic, 4-hydroxybenzoic, vanillic, trans-p-coumaric, trans-ferulic, and 3-hydroxycinnamic acids were most characteristic of Impatiens species.Additionally, various phenolic-rich extracts from leaves, flowers, and roots of Impatiens glandulifera were tested for antioxidant activity. The highest antiradical activity was detected for roots using Soxhlet extraction (EC50 = 0.055 mg [DE/ml]).The study demonstrated that members of the genus Impatiens, and in particular Impatiens glandulifera, and Impatiens noli-tangere, contain significant amounts of phenolic acids. In addition, extracts from various parts of I. glandulifera could be interesting as novel sources of natural antioxidants.     

Identification of PrP sequences essential for the interaction between the PrP polymers and Aβ peptide in a yeast-based assay

Alzheimer disease is associated with the accumulation of oligomeric amyloid β peptide (Aβ), accompanied by synaptic dysfunction and neuronal death. Polymeric form of prion protein (PrP), PrP^Sc, is implicated in transmissible spongiform encephalopathies (TSEs). Recently, it was shown that the monomeric cellular form of PrP (PrP^C), located on the neuron surface, binds Aβ oligomers (and possibly other β-rich conformers) via the PrP_23–27 and PrP_90–110 segments, acting as Aβ receptor. On the other hand, PrP^Sc polymers efficiently bind to Aβ monomers and accelerate their oligomerization. To identify specific PrP sequences that are essential for the interaction between PrP polymers and Aβ peptide, we have co-expressed Aβ and PrP (or its shortened derivatives), fused to different fluorophores, in the yeast cell. Our data show that the 90–110 and 28–89 regions of PrP control the binding of proteinase-resistant PrP polymers to the Aβ peptide, whereas the 23–27 segment of PrP is dispensable for this interaction. This indicates that the set of PrP fragments involved in the interaction with Aβ depends on PrP conformational state.     

Optimization of conditions for expression of recombinant interferon-γ in E.coli

Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.     

Development and validation of a dried blood spot—LC–APCI–MS assay for estimation of canrenone in paediatric samples

A selective and sensitive liquid chromatography (LC)–atmospheric pressure chemical ionisation (APCI)–mass spectroscopic (MS) assay of canrenone has been developed and validated employing Dried Blood Spots (DBS) as the sample collection medium. DBS samples were prepared by applying 30 μl of spiked whole blood onto Guthrie cards. A 6 mm disc was punched from the each DBS and extracted with 2 ml of methanolic solution of 17α-methyltestosterone (Internal Standard). The methanolic extract was evaporated to dryness and reconstituted in acetonitrile:water (1:9, v/v). The reconstituted solution was further subjected to solid phase extraction using HLB cartridges. Chromatographic separation was achieved using Waters Sunfire C18 reversed-phase column using isocratic elution, followed by a high organic wash to clear late eluting/highly retained components. The mobile phase consisted of methanol:water (60:40, v/v) pumped at a flow rate of 0.3 ml/min. LC–APCI–MS detection was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.1 and 303.3 for canrenone and internal standard respectively. The selectivity of the method was established by analysing DBS samples from 6 different sources (individuals). The calibration curve for canrenone was found to be linear over 25–1000 ng/ml (r > 0.994). Accuracy (% RE) and precision (% CV) values for within and between day were <20% at the lower limit of quantification (LLQC) and <15% at all other concentrations tested. The LLOQ of the method was validated at 25 ng/ml. Clinical validation of the method was achieved by employing the validated method for analysis of 160 DBS samples from 37 neonatal and paediatric patients.     

Development of a flow cytometry–based pulse-width assay for detection of aggregates in cellular therapeutics to be infused by catheter

Background aimsDelivery of cell-based therapies through the carotid artery with the use of an intra-arterial catheter could introduce aggregates and cause focal ischemia in the brain. We developed a pulse-width flow cytometry method for aggregate detection and quantification. The assay was designed to be used as a cell product release assay in a clinical trial seeking to treat ischemic stroke with sorted cells brightly expressing aldehyde dehydrogenase (ALDH^br cells) delivered through intra-arterial catheters.MethodsThe forward light scatter pulse-width axis of a flow cytometer was calibrated for particle diameter measurements through the use of traceable standard microspheres and linear regression. As a positive control, Concanavalin A–aggregated cells were counted manually and sorted onto slides to compare with pulse width–determined values. Known numbers of aggregates were spiked into purified singlet cells for quantification. A clinical standard for aggregate count and diameter was determined. The assay was used to qualify catheters with the use of ALDH^br cells.ResultsThe pulse-width axis was highly linear for microsphere diameter (r² > 0.99), which allowed for size calibration. Microscopically determined counts and diameters corresponded to pulse width-determined values. Known aggregate counts were linear with pulse width–determined aggregate counts (r² = 0.98). The limit of detection was determined to be 0.004%. Flow of ALDH^br cells through catheters did not generate aggregates. The final method to be used as a release assay for the stroke clinical trial was tested successfully on samples from volunteer donors.ConclusionsThe pulse-width aggregate detection assay provides a reliable, reproducible, accurate and rapid means of detection, classification and quantification of aggregates in cell therapy products.     

Development of an LC–MS/MS assay to determine plasma pharmacokinetics of the radioprotectant octadecyl thiophosphate (OTP) in monkeys

Octadecenyl thiophosphate (OTP), a synthetic analogue of the lysophospholipid growth factor lysophosphatidic acid (LPA), significantly reduces mortality following a lethal dose of LD_80/30 radiation exposure in a mouse model of whole-body irradiation. To facilitate dose scaling between species, we developed a novel liquid chromatography/tandem mass spectrometry (LC–MS/MS) for the preclinical pharmacokinetic characterization of OTP in monkeys. Sample extraction was carried out using a butanol based liquid–liquid extraction method. A partially deuterated OTP analogue was used as internal standard (IS). OTP and IS were separated by reversed-phase liquid chromatography on a C-8 column using 10 mM ammonium acetate and acetonitrile. A triple quadrupole mass spectrometer operating in the negative electrospray ionization mode with multiple reaction monitoring was used to detect OTP and IS transitions of m/z 363.1 → 95.0 and 403.1 → 95.0. The method was applied to determine pharmacokinetic parameters in monkeys receiving a single oral OTP dose (3 mg/kg). OTP is readily absorbed with a relatively long half-life which supports further preclinical testing of OTP as a radioprotectant in monkeys.