Structural Characterization of Monoclonal Antibodies and Epitope Mapping by FFAP Footprinting.

Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.

Shaping the future of preclinical development of successful disease-modifying drugs against Alzheimer's disease: a systematic review of tau propagation models.

The transcellular propagation of the aberrantly modified protein tau along the functional brain network is a key hallmark of Alzheimer's disease and related tauopathies. Inoculation-based tau propagation models can recapitulate the stereotypical spread of tau and reproduce various types of tau inclusions linked to specific tauopathy, albeit with varying degrees of fidelity. With this systematic review, we underscore the significance of judicious selection and meticulous functional, biochemical, and biophysical characterization of various tau inocula. Furthermore, we highlight the necessity of choosing suitable animal models and inoculation sites, along with the critical need for validation of fibrillary pathology using confirmatory staining, to accurately recapitulate disease-specific inclusions. As a practical guide, we put forth a framework for establishing a benchmark of inoculation-based tau propagation models that holds promise for use in preclinical testing of disease-modifying drugs.

Mitochondrial remodeling underlying age-induced skeletal muscle wasting: let's talk about sex.

Sarcopenia is associated with reduced quality of life and premature mortality. The sex disparities in the processes underlying sarcopenia pathogenesis, which include mitochondrial dysfunction, are ill-understood and can be decisive for the optimization of sarcopenia-related interventions. To improve the knowledge regarding the sex differences in skeletal muscle aging, the gastrocnemius muscle of young and old female and male rats was analyzed with a focus on mitochondrial remodeling through the proteome profiling of mitochondria-enriched fractions. To the best of our knowledge, this is the first study analyzing sex differences in skeletal muscle mitochondrial proteome remodeling. Data demonstrated that age induced skeletal muscle atrophy and fibrosis in both sexes. In females, however, this adverse skeletal muscle remodeling was more accentuated than in males and might be attributed to an age-related reduction of 17beta-estradiol signaling through its estrogen receptor alpha located in mitochondria. The females-specific mitochondrial remodeling encompassed increased abundance of proteins involved in fatty acid oxidation, decreased abundance of the complexes subunits, and enhanced proneness to oxidative posttranslational modifications. This conceivable accretion of damaged mitochondria in old females might be ascribed to low levels of Parkin, a key mediator of mitophagy. Despite skeletal muscle atrophy and fibrosis, males maintained their testosterone levels throughout aging, as well as their androgen receptor content, and the age-induced mitochondrial remodeling was limited to increased abundance of pyruvate dehydrogenase E1 component subunit beta and electron transfer flavoprotein subunit beta. Herein, for the first time, it was demonstrated that age affects more severely the skeletal muscle mitochondrial proteome of females, reinforcing the necessity of sex-personalized approaches towards sarcopenia management, and the inevitability of the assessment of mitochondrion-related therapeutics.

Design of AsLOV2 domain as a carrier of light-induced dissociable FMN photosensitizer.

Flavin mononucleotide (FMN) is a highly efficient photosensitizer (PS) yielding singlet oxygen (1 O2 ). However, its 1 O2 production efficiency significantly decreases upon isoalloxazine ring encapsulation into the protein matrix in genetically encoded photosensitizers (GEPS). Reducing isoalloxazine ring interactions with surrounding amino acids by protein engineering may increase 1 O2 production efficiency GEPS, but at the same time weakened native FMN-protein interactions may cause undesirable FMN dissociation. Here, in contrast, we intentionally induce the FMN release by light-triggered sulfur oxidation of strategically placed cysteines (oxidation-prone amino acids) in the isoalloxazine-binding site due to significantly increased volume of the cysteinyl side residue(s). As a proof of concept, in three variants of the LOV2 domain of Avena sativa (AsLOV2), namely V416C, T418C, and V416C/T418C, the effective 1 O2 production strongly correlated with the efficiency of irradiation-induced FMN dissociation (wild type (WT) < V416C < T418C < V416C/T418C). This alternative approach enables us: (i) to overcome the low 1 O2 production efficiency of flavin-based GEPSs without affecting native isoalloxazine ring-protein interactions and (ii) to utilize AsLOV2, due to its inherent binding propensity to FMN, as a PS vehicle, which is released at a target by light irradiation.

An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans.

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.

Isotopic Depletion Increases the Spatial Resolution of FPOP Top-Down Mass Spectrometry Analysis.

Protein radical labeling, like fast photochemical oxidation of proteins (FPOP), coupled to a top-down mass spectrometry (MS) analysis offers an alternative analytical method for probing protein structure or protein interaction with other biomolecules, for instance, proteins and DNA. However, with the increasing mass of studied analytes, the MS/MS spectra become complex and exhibit a low signal-to-noise ratio. Nevertheless, these difficulties may be overcome by protein isotope depletion. Thus, we aimed to use protein isotope depletion to analyze FPOP-oxidized samples by top-down MS analysis. For this purpose, we prepared isotopically natural (IN) and depleted (ID) forms of the FOXO4 DNA binding domain (FOXO4-DBD) and studied the protein-DNA interaction interface with double-stranded DNA, the insulin response element (IRE), after exposing the complex to hydroxyl radicals. As shown by comparing tandem mass spectra of natural and depleted proteins, the ID form increased the signal-to-noise ratio of useful fragment ions, thereby enhancing the sequence coverage by more than 19%. This improvement in the detection of fragment ions enabled us to detect 22 more oxidized residues in the ID samples than in the IN sample. Moreover, less common modifications were detected in the ID sample, including the formation of ketones and lysine carbonylation. Given the higher quality of ID top-down MSMS data set, these results provide more detailed information on the complex formation between transcription factors and DNA-response elements. Therefore, our study highlights the benefits of isotopic depletion for quantitative top-down proteomics. Data are available via ProteomeXchange with the identifier PXD044447.

Predicting nurses' safety compliance behaviour in a developing economy, using the theory of planned behaviour: A configurational approach.

AIM: The study's main objective was to use a fuzzy set qualitative comparative analysis to identify the configuration of recipes that predict nurses' safety compliance behaviour. DESIGN: A cross-sectional design. METHODS: A survey was used where questionnaires were collected from 285 nurses across four primary healthcare hospitals within the Ashanti Region, Ghana. The data collection happened between June 1 to August 2, 2022. A fuzzy set qualitative comparative analysis was used to identify the recipes of psychological factors that determine nurses' safety compliance behaviour. RESULTS: Results from the study suggest that the necessary configurations that explained nurses' safety compliance behaviour came from the presence of subjective norm, attitude, perceived behavioural control, perceived organizational support and negation of intention. The result highlights the need for safety protocols to be conscious of the interplay between nurses' assessment of self, social clues and perception of management care and support since such psychological factors must be considered concurrently to achieve the optimal safety compliance behaviour among nurses. CONCLUSION: A health and safety protocol that fails to recognize the importance of psychological antecedents on subordinates' safety compliance behaviour could limit the safety policy's usefulness in bringing the appropriate behavioural change in nurses. IMPACT: To date, no study has combined the antecedents of theory planned behaviour with perceived organizational support and cue to action to assess how they collectively predict nurses' safety compliance behaviour. Findings from the study suggest that nurses in primary health facilities inform their safety compliance behaviour by assessing self-capabilities, social signals from superiors and colleagues and perception of management support. Hospital administrators and nursing managers in sub-Saharan Africa may rely on these psychological forces to persuade nurses to develop positive safety compliance behaviour at the health facility. PATIENT OR PUBLIC CONTRIBUTION: No Patient or Public Contribution.

Quantifying the Impact of the Peptide Identification Framework on the Results of Fast Photochemical Oxidation of Protein Analysis.

Fast Photochemical Oxidation of Proteins (FPOP) is a promising technique for studying protein structure and dynamics. The quality of insight provided by FPOP depends on the reliability of the determination of the modification site. This study investigates the performance of two search engines, Mascot and PEAKS, for the data processing of FPOP analyses. Comparison of Mascot and PEAKS of the hemoglobin--haptoglobin Bruker timsTOF data set (PXD021621) revealed greater consistency in the Mascot identification of modified peptides, with around 26% of the IDs being mutual for all three replicates, compared to approximately 22% for PEAKS. The intersection between Mascot and PEAKS results revealed a limited number (31%) of shared modified peptides. Principal Component Analysis (PCA) using the peptide-spectrum match (PSM) score, site probability, and peptide intensity was applied to evaluate the results, and the analyses revealed distinct clusters of modified peptides. Mascot showed the ability to assess confident site determination, even with lower PSM scores. However, high PSM scores from PEAKS did not guarantee a reliable determination of the modification site. Fragmentation coverage of the modification position played a crucial role in Mascot assignments, while the AScore localizations from PEAKS often become ambiguous because the software employs MS/MS merging.

N6-methyladenosine in myeloid cells: a novel regulatory factor for inflammation-related diseases.

N6-methyladenosine (m6A) is one of the most abundant epitranscriptomic modifications on eukaryotic mRNA. Evidence has highlighted that m6A is altered in response to inflammation-related factors and it is closely associated with various inflammation-related diseases. Multiple subpopulations of myeloid cells, such as macrophages, dendritic cells, and granulocytes, are crucial for the regulating of immune process in inflammation-related diseases. Recent studies have revealed that m6A plays an important regulatory role in the functional of multiple myeloid cells. In this review, we comprehensively summarize the function of m6A modification in myeloid cells from the perspective of myeloid cell production, activation, polarization, and migration. Furthermore, we discuss how m6A-mediated myeloid cell function affects the progression of inflammation-related diseases, including autoimmune diseases, chronic metabolic diseases, and malignant tumors. Finally, we discuss the challenges encountered in the study of m6A in myeloid cells, intended to provide a new direction for the study of the pathogenesis of inflammation-related diseases.

Autosomal dominant ApoA4 mutations present as tubulointerstitial kidney disease with medullary amyloidosis.

Sporadic cases of apolipoprotein A-IV medullary amyloidosis have been reported. Here we describe five families found to have autosomal dominant medullary amyloidosis due to two different pathogenic APOA4 variants. A large family with autosomal dominant chronic kidney disease (CKD) and bland urinary sediment underwent whole genome sequencing with identification of a chr11:116692578 G>C (hg19) variant encoding the missense mutation p.L66V of the ApoA4 protein. We identified two other distantly related families from our registry with the same variant and two other distantly related families with a chr11:116693454 C>T (hg19) variant encoding the missense mutation p.D33N. Both mutations are unique to affected families, evolutionarily conserved and predicted to expand the amyloidogenic hotspot in the ApoA4 structure. Clinically affected individuals suffered from CKD with a bland urinary sediment and a mean age for kidney failure of 64.5 years. Genotyping identified 48 genetically affected individuals; 44 individuals had an estimated glomerular filtration rate (eGFR) under 60 ml/min/1.73 m2, including all 25 individuals with kidney failure. Significantly, 11 of 14 genetically unaffected individuals had an eGFR over 60 ml/min/1.73 m2. Fifteen genetically affected individuals presented with higher plasma ApoA4 concentrations. Kidney pathologic specimens from four individuals revealed amyloid deposits limited to the medulla, with the mutated ApoA4 identified by mass-spectrometry as the predominant amyloid constituent in all three available biopsies. Thus, ApoA4 mutations can cause autosomal dominant medullary amyloidosis, with marked amyloid deposition limited to the kidney medulla and presenting with autosomal dominant CKD with a bland urinary sediment. Diagnosis relies on a careful family history, APOA4 sequencing and pathologic studies.

Revealing the nuclearity of iron citrate complexes at biologically relevant conditions.

Citric acid plays an ubiquitous role in the complexation of essential metals like iron and thus it has a key function making them biologically available. For this, iron(III) citrate complexes are considered among the most significant coordinated forms of ferric iron that take place in biochemical processes of all living organisms. Although these systems hold great biological relevance, their coordination chemistry has not been fully elucidated yet. The current study aimed to investigate the speciation of iron(III) citrate using Mössbauer and electron paramagnetic resonance spectroscopies. Our aim was to gain insights into the structure and nuclearity of the complexes depending on the pH and iron to citrate ratio. By applying the frozen solution technique, the results obtained directly reflect the iron speciation present in the aqueous solution. At 1:1 iron:citrate molar ratio, polynuclear species prevailed forming most probably a trinuclear structure. In the case of citrate excess, the coexistence of several monoiron species with different coordination environments was confirmed. The stability of the polynuclear complexes was checked in the presence of organic solvents.

Efficacy assessment of an active tau immunotherapy in Alzheimer's disease patients with amyloid and tau pathology: a post hoc analysis of the "ADAMANT" randomised, placebo-controlled, double-blind, multi-centre, phase 2 clinical trial.

BACKGROUND: Tau pathology correlates with and predicts clinical decline in Alzheimer's disease. Approved tau-targeted therapies are not available. METHODS: ADAMANT, a 24-month randomised, placebo-controlled, parallel-group, double-blinded, multicenter, Phase 2 clinical trial (EudraCT2015-000630-30, NCT02579252) enrolled 196 participants with Alzheimer's disease; 119 are included in this post-hoc subgroup analysis. AADvac1, active immunotherapy against pathological tau protein. A machine learning model predicted likely Amyloid+Tau+ participants from baseline MRI. STATISTICAL METHODS: MMRM for change from baseline in cognition, function, and neurodegeneration; linear regression for associations between antibody response and endpoints. RESULTS: The prediction model achieved PPV of 97.7% for amyloid, 96.2% for tau. 119 participants in the full analysis set (70 treatment and 49 placebo) were classified as A+T+. A trend for CDR-SB 104-week change (estimated marginal means [emm] = -0.99 points, 95% CI [-2.13, 0.13], p = 0.0825]) and ADCS-MCI-ADL (emm = 3.82 points, CI [-0.29, 7.92], p = 0.0679) in favour of the treatment group was seen. Reduction was seen in plasma NF-L (emm = -0.15 log pg/mL, CI [-0.27, -0.03], p = 0.0139). Higher antibody response to AADvac1 was related to slowing of decline on CDR-SB (rho = -0.10, CI [-0.21, 0.01], p = 0.0376) and ADL (rho = 0.15, CI [0.03, 0.27], p = 0.0201), and related to slower brain atrophy (rho = 0.18-0.35, p < 0.05 for temporal volume, whole cortex, and right and left hippocampus). CONCLUSIONS: In the subgroup of ML imputed or CSF identified A+T+, AADvac1 slowed AD-related decline in an antibody-dependent manner. Larger anti-tau trials are warranted. FUNDING: AXON Neuroscience SE.

Safety and Performance of Narhinel 0.9% Sodium Chloride Monodose and Otrisal 0.74% Sodium Chloride Monodose Nasal Saline Solutions and Nasal Aspirators in Real-World Settings: Postmarket Clinical Follow-up Study Results.

Background: Blocked or stuffy nose is a common and bothersome symptom of colds, particularly for young children who are unable to clear their noses on their own. Nasal saline solutions and nasal aspirators are designed to gently cleanse and remove blocking nasal secretions. Objective: To assess the safety and performance of 2 monodose isotonic saline solutions (Narhinel 0.9% and Otrisal 0.74% sodium chloride; GSK Consumer Healthcare SARL, a Haleon company, Nyon, Switzerland) and 2 nasal aspirators with disposable hard- and soft-nozzle refills used as a standalone or combination treatment. Methods: We conducted 2 observational, online questionnaire-based, postmarket clinical follow-up studies in Europeans who had used any of the devices ≥1 time in the past 6 months. Coprimary objectives were to confirm the safety and performance of the saline solutions (Narhinel and Otrisal, Study 1) and nasal aspirators (with hard- and soft-nozzle refills, Study 2). Safety was assessed via the proportion of patients reporting adverse events and/or device malfunctions while using the devices within the previous 6 months, and performance was assessed by satisfaction rated on a 5-point scale, with "satisfied" and "very satisfied" being the highest performance ratings. Results: A total of 1136 (Study 1) and 1237 (Study 2) questionnaires were initiated by volunteer participants. Less than 2% of participants reported adverse events for any evaluated product in the previous 6 months. Most participants were "satisfied" or "very satisfied" with the devices for their intended use, with 78% to 91% of participants in the Narhinel arm, 73%-94% in the Otrisal arm, 71% to 95% in the soft-nozzle arm, and 71% to 80% in the hard-nozzle arm giving these ratings. Conclusions: These data support the safety and performance of 2 monodose saline solutions (Narhinel and Otrisal) for nasal cleansing, nasal moisturization, and/or loosening nasal secretions, and of nasal aspirators (with hard- and soft-nozzle refills) for clearing a blocked nose and removing nasal secretions.

Sensitivity of Individual and Composite Test Scores from the Cogstate Brief Battery to Mild Cognitive Impairment and Dementia Due to Alzheimer's Disease.

BACKGROUND: The Cogstate Brief Battery (CBB) is a computerized cognitive test battery used commonly to identify cognitive deficits related to Alzheimer's disease (AD). However, AD and normative samples used to understand the sensitivity of the CBB to AD in the clinic have been limited, as have the outcome measures studied. OBJECTIVE: This study investigated the sensitivity of CBB outcomes, including potential composite scores, to cognitive impairment in mild cognitive impairment (MCI) and dementia due to AD, in carefully selected samples. METHODS: Samples consisted of 4,871 cognitively unimpaired adults and 184 adults who met clinical criteria for MCI (Clinical Dementia Rating (CDR) = 0.5) or dementia (CDR > 0.5) due to AD and CBB naive. Speed and accuracy measures from each test were examined, and theoretically- and statistically-derived composites were created. Sensitivity and specificity of classification of cognitive impairment were compared between outcomes. RESULTS: Individual CBB measures of learning and working memory showed high discriminability for AD-related cognitive impairment for CDR 0.5 (AUCs ∼ 0.79-0.88), and CDR > 0.5 (AUCs ∼ 0.89-0.96) groups. Discrimination ability for theoretically derived CBB composite measures was high, particularly for the Learning and Working Memory (LWM) composite (CDR 0.5 AUC = 0.90, CDR > 0.5 AUC = 0.97). As expected, statistically optimized linear composite measures showed strong discrimination abilities albeit similar to the LWM composite. CONCLUSIONS: In older adults, the CBB is effective for discriminating cognitive impairment due to MCI or AD-dementia from unimpaired cognition with the LWM composite providing the strongest sensitivity.

Top-Down Proteoform Analysis by 2D MS with Quadrupolar Detection.

Two-dimensional mass spectrometry (2D MS) is a multiplexed tandem mass spectrometry method that does not rely on ion isolation to correlate the precursor and fragment ions. On a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS), 2D MS instead uses the modulation of precursor ion radii inside the ICR cell before fragmentation and yields 2D mass spectra that show the fragmentation patterns of all the analytes. In this study, we perform 2D MS for the first time with quadrupolar detection in a dynamically harmonized ICR cell. We discuss the advantages of quadrupolar detection in 2D MS and how we adapted existing data processing techniques for accurate frequency-to-mass conversion. We apply 2D MS with quadrupolar detection to the top-down analysis of covalently labeled ubiquitin with ECD fragmentation, and we develop a workflow for label-free relative quantification of biomolecule isoforms in 2D MS.

Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS.

Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins, and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins.

Probing Antibody Structures by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation. Despite these difficulties, there are already numerous excellent studies describing MS-based antibody structure characterization. In this chapter, we describe a universal HDX-MS workflow. Deeper attention is paid to sample handling, optimization procedures, and feasibility stages, as these elements of the HDX experiment are crucial for obtaining reliable detailed and spatially well-resolved information.

Multivalency of nucleosome recognition by LEDGF.

Eukaryotic transcription is dependent on specific histone modifications. Their recognition by chromatin readers triggers complex processes relying on the coordinated association of transcription regulatory factors. Although various modification states of a particular histone residue often lead to differential outcomes, it is not entirely clear how they are discriminated. Moreover, the contribution of intrinsically disordered regions outside of the specialized reader domains to nucleosome binding remains unexplored. Here, we report the structures of a PWWP domain from transcriptional coactivator LEDGF in complex with the H3K36 di- and trimethylated nucleosome, indicating that both methylation marks are recognized by PWWP in a highly conserved manner. We identify a unique secondary interaction site for the PWWP domain at the interface between the acidic patch and nucleosomal DNA that might contribute to an H3K36-methylation independent role of LEDGF. We reveal DNA interacting motifs in the intrinsically disordered region of LEDGF that discriminate between the intra- or extranucleosomal DNA but remain dynamic in the context of dinucleosomes. The interplay between the LEDGF H3K36-methylation reader and protein binding module mediated by multivalent interactions of the intrinsically disordered linker with chromatin might help direct the elongation machinery to the vicinity of RNA polymerase II, thereby facilitating productive elongation.

[A case of botulism in the Czech Republic and current possibilities for detecting the neurotoxin produced by Clostridium botulinum].

In the Czech Republic, botulism is a rare life-threatening disease. A total of 155 cases have been reported since 1960; according to the ISIN (formerly EPIDAT) database, there have been only three isolated cases since 2013, with the exception of a single occurrence of familial botulism in 2013. In our work, we present the occurrence of botulism after ingestion of pâté of untraceable origin by a couple who were hospitalized for botulotoxin food poisoning in July 2022. Their neurological symptoms were dominated by dysarthria. After administration of antibotulinum serum, their condition improved significantly. Patient samples were analyzed using affinity carriers and MALDI mass spectrometry, a modern highly sensitive technique for detecting the presence of botulinum neurotoxins. Unlike traditional detection by a difficult and costly biological experiment on mice, the above analysis does not require the killing of laboratory animals.