In this publication, we report on the development of a quantitative enzymatic method for the detection of four botulinum neurotoxin (BoNT) serotypes responsible for human botulism by MALDI-TOF mass spectrometry. Factors that might affect the linearity and dynamic range for detection of BoNT cleavage products were initially examined, including the amount of peptide substrate and internal standard, the timing of cleavage reaction, and the components in the reaction solution. It was found that a long incubation time produced sensitive results, but was not capable of determining higher toxin concentrations, whereas a short incubation time was less sensitive so that lower toxin concentrations were not detected. In order to overcome these limitations, a two-stage analysis strategy was applied. The first stage analysis involved a short incubation period (e.g., 30 min). If no toxin was detected at this stage, the cleavage reaction was allowed to continue and the samples were analyzed at a second time point (4 h), so that toxin levels lower than 1 mouse LD50 or 55 attomoles per milliliter (55 amol/mL) could be quantified. By combining the results from two-stage quantification, 4 or 5 orders of magnitude in dynamic range were achieved for the detection of the serotypes of BoNT/A, BoNT/B, BoNT/E, or BoNT/F. The effect of multiplexing the assay by mixing substrates for different BoNT serotypes into a single reaction was also investigated in order to reduce the numbers of the cleavage reactions and save valuable clinical samples.
Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances known to mankind. It is essential to have a simple, quick and sensitive method for the detection and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory has developed a mass spectrometry-based Endopep-MS assay that is able to rapidly detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of seven distinctive BoNT serotypes (A to G) and is the causative agent of botulism in both humans and animals. To improve the sensitivity of the Endopep-MS assay, we report here the development of novel peptide substrates for the detection of BoNT/E activity through systematic and comprehensive approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement in sensitivity compared to the current substrate for the detection of both not trypsin-activated and trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouseLD50/mL was achieved using the novel peptide substrate in the assay to detect not trypsin-activated BoNT/E complex spiked in serum, stool and food samples.
Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.
Clostridium botulinum neurotoxins (BoNTs) cause the life-threatening disease botulism through the inhibition of neurotransmitter release by cleaving essential SNARE proteins. There are seven serologically distinctive types of BoNTs and many subtypes within a serotype have been identified. BoNT/A5 is a recently discovered subtype of type A botulinum neurotoxin which possesses a very high degree of sequence similarity and identity to the well-studied A1 subtype. In the present study, we examined the endopeptidase activity of these two BoNT/A subtypes and our results revealed significant differences in substrate binding and cleavage efficiency between subtype A5 and A1. Distinctive hydrolysis efficiency was observed between the two toxins during cleavage of the native substrate SNAP-25 versus a shortened peptide mimic. N-terminal truncation studies demonstrated that a key region of the SNAP-25, including the amino acid residues at 151 through 154 located in the remote binding region of the substrate, contributed to the differential catalytic properties between A1 and A5. Elevated binding affinity of the peptide substrate resulted from including these important residues and enhanced BoNT/A5s hydrolysis efficiency. In addition, mutations of these amino acid residues affect the proteolytic performance of the two toxins in different ways. This study provides a better understanding of the biological activity of these toxins, their performance characteristics in the Endopep-MS assay to detect BoNT in clinical samples and foods, and is useful for the development of peptide substrates.
Background: Antibody-drug conjugates (ADCs) combine the characteristics of large-molecule biologics and small-molecule drugs and are heterogeneous mixtures that can biotransform in vivo, resulting in additional complexity. ADC bioanalytical strategies require novel analytical methods, as well as existing large- and small-molecule methods. Because ADCs in late-stage clinical development are relatively new, regulatory guidelines and standard industry best practices for developing strategies for bioanalytical PK assays are still being established. Results: A PK assay strategy was developed that included comprehensive novel reagent and assay characterization approaches for the ADC ado-trastuzumab emtansine (T-DM1). Conclusion: The bioanalytical strategy was successfully applied to the drug development of T-DM1 and ensured that key analytes were accurately measured in support of nonclinical and clinical development.
Botulinum neurotoxins (BoNT) are the deadliest agents known. Previously, we reported an endopeptidase activity based method (Endopep-MS) that detects and differentiates BoNT serotypes A-G. This method uses serotype specific monoclonal antibodies and the specific enzymatic activity of BoNT against peptide substrates which mimic the toxins natural target. Cleavage products from the reaction are detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We have now developed a multiple reaction monitoring method to quantify the biological activity of BoNT serotypes A (BoNT/A) and B (BoNT/B) present in 0.5 mL of serum using electrospray mass spectrometry. The limit of quantification for each serotype is 1 mouse intraperitoneal lethal dose (MIPLD(50)) corresponding to 31 pg of BoNT/A and 15 pg of BoNT/B in this study. This method was applied to serum from rhesus macaques with inhalational botulism following exposure to BoNT/B, showing a maximum activity of 6.0 MIPLD(50)/mL in surviving animals and 653.6 MIPLD(50)/mL in animals that died in the study. The method detects BoNT/B in serum 2-5 h after exposure and up to 14 days. This is the first report of a quantitative method with sufficient sensitivity, selectivity, and low sample size requirements to measure circulating BoNT activity at multiple times during the course of botulism.
Botulinum neurotoxins (BoNTs) cause botulism by cleaving proteins necessary for nerve transmission. There are seven serotypes of BoNT, A-G, characterized by their response to antisera. Many serotypes are further distinguished into differing subtypes based on amino acid sequence, some of which result in functional differences. Our laboratory previously reported that all tested subtypes within each serotype have the same site of enzymatic activity. Recently, three new subtypes of BoNT/F; /F3, /F4, and /F5, were reported. Here, we report that BoNT/F5 cleaves substrate synaptobrevin-2 in a different location than the other BoNT/F subtypes, between (54)L and (55)E. This is the first report of cleavage of synaptobrevin-2 in this location.
Botulinum neurotoxins (BoNTs) are the most toxic substances known to humankind. Rapid and sensitive detection of BoNTs is necessary for timely clinical confirmation of the disease state in botulism. BoNTs cleave proteins and peptide mimics at specific sites. A mass spectrometry (MS)-based method, Endopep-MS, can detect these cleavages and has detection limits of 0.05-0.5 mouse LD(50) (U) in serum, depending on the BoNT serotypes. In this method, the products generated from cleavage of peptide substrates using antibody affinity-purified toxins are detected by MS. Nonspecific bound endogenous proteases or peptidases in stool can coextract with the toxin, cleaving the peptide substrates and reducing the sensitivity of the method. Here we report a method to reduce nonspecific substrate cleavage by reducing stool protease coextraction in the Endopep-MS assay.
Antibody-drug conjugates (ADCs) are designed to facilitate the targeted delivery of cytotoxic drugs to improve their tumor fighting effects and minimize systemic toxicity. However, efficacy and safety can potentially be compromised due to the release of conjugated drugs from the ADC with time while in circulation, resulting in changes in the drug-to-antibody ratio (DAR). Current understanding of this process is limited because existing methods such as immunoassays fail to distinguish ADCs with different DARs. Here we demonstrate a novel method with bead-based affinity capture and capillary liquid chromatography-mass spectrometry to allow direct measurement of drug release by quantifying DAR distributions of the ADC in plasma/serum. This method successfully identified individual intact conjugated antibody species produced due to drug loss from ADCs (e.g., an engineered site-specific anti-MUC16 THIOMAB-drug conjugate) and measured the corresponding DAR distributions in vitro and in vivo. Information obtained can provide insights into the mechanisms involved in drug loss and help to optimize ADC therapeutics. Other potential applications of the method may include characterization of posttranslational modifications, protein adducts, and immunogenicity.
Botulinum neurotoxins (BoNTs) cause botulism, which can be fatal if it is untreated. BoNTs cleave proteins necessary for nerve transmission, resulting in paralysis. The in vivo protein target has been reported for all seven serotypes of BoNT, i.e., serotypes A to G. Knowledge of the cleavage sites has led to the development of several assays to detect BoNT based on its ability to cleave a peptide substrate derived from its in vivo protein target. Most serotypes of BoNT can be subdivided into subtypes, and previously, we demonstrated that three of the currently known subtypes of BoNT/F cleave a peptide substrate, a shortened version of synaptobrevin-2, between Q58 and K59. However, our research indicated that Clostridium baratii type F toxin did not cleave this peptide. In this study, we detail experiments demonstrating that Clostridium baratii type F toxin cleaves recombinant synaptobrevin-2 in the same location as that cleaved by proteolytic F toxin. In addition, we demonstrate that Clostridium baratii type F toxin can cleave a peptide substrate based on the sequence of synaptobrevin-2. This peptide substrate is an N-terminal extension of the original peptide substrate used for detection of other BoNT/F toxins and can be used to detect four of the currently known BoNT/F subtypes by mass spectrometry.
Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing respiratory failure leading to long-term intensive care or death. The best treatment for botulism includes serotype-specific antitoxins, which are most effective when administered early in the course of the intoxication. Early confirmation of human exposure to any serotype of BoNT is an important public health goal. In previous work, we focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating the seven serotypes (BoNT/A-G) in buffer and BoNT/A, /B, /E, and /F (the four serotypes that commonly affect humans) in clinical samples. We have previously reported the success of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. However, to check for any one of the four serotypes of BoNT/A, /B, /E, or /F, each sample is split into 4 aliquots, and tested for the specific serotypes separately. The discovery of a unique monoclonal antibody that recognizes all four serotypes of BoNT/A, /B, /E and /F allows us to perform simultaneous detection of all of them. When applied in conjunction with the Endopep-MS assay, the detection limit for each serotype of BoNT with this multi-specific monoclonal antibody is similar to that obtained when using other serotype-specific antibodies.
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.
Botulinum neurotoxin (BoNT) is one of the most toxic substances known. BoNT is classified into seven distinct serotypes labeled A-G. Among individual serotypes, researchers have identified subtypes based on amino acid variability within a serotype and toxin variants with minor amino acid sequence differences within a subtype. BoNT subtype identification is valuable for tracing and tracking bacterial pathogens. A proteomics approach is useful for BoNT subtyping since botulism is caused by botulinum neurotoxin and does not require the presence of the bacteria or its DNA. Enzymatic digestion and peptide identification using tandem mass spectrometry determines toxin protein sequences. However, with the conventional one-step digestion method, producing sufficient numbers of detectable peptides to cover the entire protein sequence is difficult, and incomplete sequence coverage results in uncertainty in distinguishing BoNT subtypes and toxin variants because of high sequence similarity. We report here a method of multiple enzymes and sequential in-gel digestion (MESID) to characterize the BoNT protein sequence. Complementary peptide detection from toxin digestions has yielded near-complete sequence coverage for all seven BoNT serotypes. Application of the method to a BoNT-contaminated carrot juice sample resulted in the identification of 98.4% protein sequence which led to a confident determination of the toxin subtype.
Botulinum neurotoxins (BoNTs) cause the disease botulism, which can be lethal if untreated. There are seven known serotypes of BoNT, A-G, defined by their response to antisera. Many serotypes are distinguished into differing subtypes based on amino acid sequence, and many subtypes are further differentiated into toxin variants. Previous work in our laboratory described the use of a proteomics approach to distinguish subtype BoNT/A1 from BoNT/A2 where BoNT identities were confirmed after searching data against a database containing protein sequences of all known BoNT/A subtypes. We now describe here a similar approach to differentiate subtypes BoNT/B1, /B2, /B3, /B4, and /B5. Additionally, to identify new subtypes or hitherto unpublished amino acid substitutions, we created an amino acid substitution database covering every possible amino acid change. We used this database to differentiate multiple toxin variants within subtypes of BoNT/B1 and B2. More importantly, with our amino acid substitution database, we were able to identify a novel BoNT/B subtype, designated here as BoNT/B7. These techniques allow for subtype and strain level identification of both known and unknown BoNT/B rapidly with no DNA required.
The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
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