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Cyclodepsipeptides (CDPs) represent a huge family of chemically and structurally diverse molecules with a wide ability for molecular interactions. CDPs are cyclic peptide-related natural products made up of both proteinogenic and nonproteinogenic amino acids linked by amide and ester bonds. The combined use of different analytical methods is required to accurately determine their integral structures including stereochemistry, thus allowing deeper insights into their often-intriguing bioactivities and their possible usefulness. Our goal is to present the various methods developed to accurately characterize CDPs. Presently, Marfey's method and NMR (nuclear magnetic resonance) are still considered the best for characterizing CDP configuration. Nevertheless, electrospray-high resolution tandem mass spectrometry (ESI-HRMS/MS) is of great value for efficiently resolving CDP's composition and sequences. For instance, recent data shows that the fragmentation of cationized CDPs (e.g., [M + Li]+ and [M + Na]+) leads to selective cleavage of ester bonds and specific cationized product ions (b series) useful to get unprecedented sequence information. Thus, after a brief presentation of their structure, biological functions, and biosynthesis, we also provide a historic overview of these various analytical approaches as well as their advantages and limitations with a special emphasis on the emergence of methods based on HRMS/MS through recent fundamental works and applications.
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Ricin is a toxic protein regarded as a potential chemical weapon for bioterrorism or criminal use. In the event of a ricin incident, rapid analytical methods are essential for ricin confirmation in a diversity of matrices, from environmental to human or food samples. Mass spectrometry-based methods provide specific toxin identification but require prior enrichment by antibodies to reach trace-level detection in matrices. Here, we describe a novel assay using the glycoprotein asialofetuin as an alternative to antibodies for ricin enrichment, combined with the specific detection of signature peptides by high-resolution mass spectrometry. Additionally, optimizations made to the assay reduced the sample preparation time from 5 h to 80 min only. Method evaluation confirmed the detection of ricin at trace levels over a wide range of pH and in protein-rich samples, illustrating challenging matrices. This new method constitutes a relevant antibody-free solution for the fast and specific mass spectrometry detection of ricin in the situation of a suspected toxin incident, complementary to active ricin determination by adenine release assays.
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In March 2023, 34 associated cases of iatrogenic botulism were detected in Germany (30 cases), Switzerland (two cases), Austria (one case), and France (one case). An alert was rapidly disseminated via European Union networks and communication platforms (Food- and Waterborne Diseases and Zoonoses Network, EpiPulse, Early Warning and Response System) and the International Health Regulation mechanism; the outbreak was investigated in a European collaboration. We traced sources of the botulism outbreak to treatment of weight loss in Türkiye, involving intragastric injections of botulinum neurotoxin. Cases were traced using a list of patients who had received this treatment. Laboratory investigations of the first 12 German cases confirmed nine cases. The application of innovative and highly sensitive endopeptidase assays was necessary to detect minute traces of botulinum neurotoxin in patient sera. The botulism notification requirement for physicians was essential to detect this outbreak in Germany. The surveillance case definition of botulism should be revisited and inclusion of cases of iatrogenic botulism should be considered as these cases might lack standard laboratory confirmation yet warrant public health action. Any potential risks associated with the use of botulinum neurotoxins in medical procedures need to be carefully balanced with the expected benefits of the procedure.
Assuntos
Toxinas Botulínicas , Botulismo , Clostridium botulinum , Animais , Humanos , Toxinas Botulínicas/efeitos adversos , Botulismo/diagnóstico , Botulismo/epidemiologia , Botulismo/etiologia , Neurotoxinas , Viagem , Surtos de Doenças , Redução de Peso , Doença Iatrogênica/epidemiologiaRESUMO
We addressed here the need for improved sensitivity of top-down mass spectrometry for identification, differentiation, and absolute quantification of sequence variants of SEA, a bacterial toxin produced by Staphylococcus aureus and regularly involved in food poisoning outbreaks (FPO). We combined immunoaffinity enrichment, a protein internal standard, and optimized acquisition conditions, either by full-scan high-resolution mass spectrometry (HRMS) or multiplex parallel reaction monitoring (PRM) mode. Deconvolution of full-scan HRMS signal and PRM detection of variant-specific fragment ions allowed confident identification of each SEA variant. Summing the PRM signal of variant-common fragment ions was most efficient for absolute quantification, illustrated by a sensitivity down to 2.5 ng/mL and an assay variability below 15%. Additionally, we showed that relative PRM fragment ion abundances constituted a supplementary specificity criterion in top-down quantification. The top-down method was successfully evaluated on a panel of enterotoxin-producing strains isolated during FPO, in parallel to the conventional whole genome sequencing, ELISA, and bottom-up mass spectrometry methods. Top-down provided at the same time correct identification of the SEA variants produced and precise determination of the toxin level. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01710.
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Enterotoxinas , Microbiologia de Alimentos , Enterotoxinas/análise , Enterotoxinas/genética , Enterotoxinas/metabolismo , Espectrometria de Massas/métodos , Staphylococcus aureus/metabolismoRESUMO
Alternative methods to RT-PCR for SARS-CoV-2 detection are investigated to provide complementary data on viral proteins, increase the number of tests performed, or identify false positive/negative results. Here, we have developed a simple mass spectrometry assay for SARS-CoV-2 in nasopharyngeal swab samples using common laboratory reagents. The method employs high sensitivity and selectivity targeted mass spectrometry detection, monitoring nine constitutive peptides representative of the three main viral proteins and a straightforward pellet digestion protocol for convenient routine applications. Absolute quantification of N, M, and S proteins was achieved by addition of isotope-labeled versions of best peptides. Limit of detection, recovery, precision, and linearity were thoroughly evaluated in four representative viral transport media (VTM) containing distinct total protein content. The protocol was sensitive in all swab media with limit of detection determined at 2 × 103 pfu/mL, corresponding to as low as 30 pfu injected into the LC-MS/MS system. When tested on VTM-stored nasopharyngeal swab samples from positive and control patients, sensitivity was similar to or better than rapid immunoassay dipsticks, revealing a corresponding RT-PCR detection threshold at Ct â¼ 24. The study represents the first thorough evaluation of sensitivity and robustness of targeted mass spectrometry in nasal swabs, constituting a promising SARS-CoV-2 antigen assay for the first-line diagnosis of COVID-19 and compatible with the constraints of clinical settings. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01646.
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COVID-19/diagnóstico , Cromatografia Líquida/métodos , Nasofaringe/virologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Espectrometria de Massas em Tandem/métodos , COVID-19/virologia , Meios de Cultura , Humanos , Nucleocapsídeo/metabolismo , Proteômica/métodos , Reprodutibilidade dos Testes , SARS-CoV-2/fisiologia , Sensibilidade e Especificidade , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Lethal and edema toxins are critical virulence factors of Bacillus anthracis. Few data are available on their presence in the early stage of intranasal infection. METHODS: To investigate the diffusion of edema factor (EF) and lethal factor (LF), we use sensitive quantitative methods to measure their enzymatic activities in mice intranasally challenged with a wild-type B anthracis strain or with an isogenic mutant deficient for the protective antigen. RESULTS: One hour after mouse challenge, although only 7% of mice presented bacteremia, LF and EF were detected in the blood of 100% and 42% of mice, respectively. Protective antigen facilitated the diffusion of LF and EF into the blood compartment. Toxins played a significant role in the systemic dissemination of B anthracis in the blood, spleen, and liver. A mouse model of intoxination further confirmed that LT and ET could diffuse rapidly in the circulation, independently of bacteria. CONCLUSIONS: In this inhalational model, toxins have disseminated rapidly in the blood, playing a significant and novel role in the early systemic diffusion of bacteria, demonstrating that they may represent a very early target for the diagnosis and the treatment of anthrax.
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Antraz/metabolismo , Antígenos de Bactérias/sangue , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/sangue , Absorção Nasal , Fatores de Virulência/sangue , Animais , Animais não Endogâmicos , Antraz/microbiologia , Bacillus anthracis/enzimologia , Bacteriemia , Biomarcadores/sangue , Modelos Animais de Doenças , Ativação Enzimática , Ensaios Enzimáticos , Feminino , Camundongos , VirulênciaRESUMO
Well-characterized prognostic biomarkers and reliable quantitative methods are key in sepsis management. Among damage-associated molecular patterns, S100A8/S100A9 complexes are reported to be markers for injured cells and to improve the prediction of death in septic shock patients. In view of the structural diversity observed for the intracellular forms, insight into circulating complexes and proteoforms is required to establish prognostic biomarkers. Here, we developed top-down and bottom-up proteomics to characterize the association of S100A8 and S100A9 in complexes and major circulating proteoforms. An antibody-free method was developed for absolute quantification of S100A8/S100A9 in a cohort of 49 patients to evaluate the prognostic value on the first day after admission for septic shock. The predominant circulating forms identified by top-down proteomics were S100A8, mono-oxidized S100A8, truncated acetylated S100A9, and S-nitrosylated S100A9. S100A8, truncated acetylated S100A9, and mono-oxidized S100A8 discriminated between survivors and nonsurvivors, along with total S100A8/S100A9 measured by the antibody-free bottom-up method. Overall, new insights into circulating S100A8/S100A9 and confirmation of its prognostic value in septic shock are crucial in qualification of this biomarker. Also, the simple antibody-free assay would support the harmonization of S100A8/S100A9 measurements.
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Proteômica , Choque Séptico , Calgranulina A/genética , Calgranulina B/genética , Humanos , Prognóstico , Choque Séptico/diagnósticoRESUMO
AIMS: Cetuximab associated with cisplatin and 5-fluorouracil is used to treat patients with inoperable or metastatic head and neck squamous cell carcinomas (HNSCC) up until disease progression or unacceptable toxicities. To date, no biomarkers of efficacy are available to select patients who will benefit from treatment. METHODS: An ancillary pharmacokinetics (PK) exploration was performed in the context of a prospective study investigating circulating-tumour cells vs progression-free survival (PFS). Cetuximab plasma concentrations were analysed according to a population PK model. Individual exposure parameters were confronted with soluble epidermal growth factor receptor (sEGFR) concentrations, tumour response and PFS. RESULTS: PK data (28 patients, 203 observations) were best described by a two-compartment model with linear elimination. Performance status (PS) significantly correlated to both cetuximab clearance and central volume of distribution with both parameters increasing by 33.3% (95% CI 1-65.6) for each 1-point increase of PS compared to PS = 0. Univariate analysis showed that patients with higher trough cetuximab concentrations at Day 7 (Cmin,D7 ) had better tumour response (P = 0.03) and longer PFS (P = 0.035). However, multivariate analysis revealed that only PS and tumour size at baseline remained significantly associated with PFS. Levels of sEGFR increased during cetuximab treatment but were not associated with PFS in the multivariate analysis. CONCLUSIONS: Our study prospectively indicates that PS is likely a confounding factor in the relationship between cetuximab PK and PFS, patients with a poor PS having lower cetuximab plasma exposure and lower PFS.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Cetuximab/farmacocinética , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Modelos Biológicos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores Tumorais/sangue , Cetuximab/administração & dosagem , Cetuximab/efeitos adversos , Cisplatino/administração & dosagem , Cisplatino/farmacocinética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/sangue , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/farmacocinética , Neoplasias de Cabeça e Pescoço/sangue , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/efeitos dos fármacos , Células Neoplásicas Circulantes/patologia , Intervalo Livre de Progressão , Estudos Prospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço/sangue , Carcinoma de Células Escamosas de Cabeça e Pescoço/diagnóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Adulto JovemRESUMO
Tau and α-synuclein are central in several neurodegenerative diseases, including Alzheimer Disease (AD), Dementia with Lewy Bodies (DLB) and Parkinson Disease (PD). New analytical methods for precise quantification of cerebrospinal fluid (CSF) levels of both tau and α-synuclein are required to differentiate between dementias or monitor therapeutic responses. Notably, levels of total α-synuclein reported by ELISA are inconsistent among studies, impacted by antibody specificity or lack of standardization. Here, we report on the development and validation of a sensitive and robust mass spectrometry-based assay for the simultaneous quantification of tau and α-synuclein in CSF. The optimized workflow avoided any affinity reagents, and involved the combination of two enzymes, Glu-C and trypsin for optimal sequence coverage of α-synuclein acidic C-terminus. Up to 7 α-synuclein peptides were quantified, including the C-terminal peptide (132-140), resulting in a sequence coverage of 54% in CSF. The lower limits of quantification (LLOQ) ranged from 0.1 ng mL-1 to 1 ng mL-1 depending on the peptide. Regarding CSF tau, 4 peptides common to all isoforms were monitored, and LLOQ ranged from 0.5 ng mL-1 to 0.75 ng mL-1. The multiplex method was successfully applied to CSF samples from AD and DLB patients, two clinically overlapping neurodegenerative diseases. CSF α-synuclein levels were significantly lower in DLB patients compared to AD and controls. Moreover, tau and α-synuclein concentrations showed opposite trends in AD and DLB patients, suggesting the benefit of combining the two biomarkers for differentiation of DLB from AD and controls.
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Doença de Alzheimer/diagnóstico , Doença por Corpos de Lewy/diagnóstico , alfa-Sinucleína/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Sequência de Aminoácidos , Biomarcadores/líquido cefalorraquidiano , Cromatografia Líquida , Diagnóstico Diferencial , Humanos , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteólise , Serina Endopeptidases/química , Espectrometria de Massas em Tandem , Tripsina/química , alfa-Sinucleína/química , Proteínas tau/químicaRESUMO
The treatment of Alzheimer's disease (AD) remains challenging and requires a better in depth understanding of AD progression. Particularly, the link between amyloid protein precursor (APP) processing and Tau pathology development remains poorly understood. Growing evidences suggest that APP processing and amyloid-ß (Aß) release are upstream of Tau pathology but the lack of animal models mimicking the slow progression of human AD raised questions around this mechanism. Here, we described that an AD-like ßAPP processing in adults wild-type rats, yielding to human APP, ßCTF and Aß levels similar to those observed in AD patients, is sufficient to trigger gradual Tauopathy. The Tau hyperphosphorylation begins several months before the formation of both amyloid plaques and tangle-like aggregates in aged rats and without associated inflammation. Based on a longitudinal characterization over 30 months, we showed that extrasynaptic and emotional impairments appear before long-term potentiation deficits and memory decline and so before Aß and Tau aggregations. These compelling data allowed us to (1) experimentally confirm the causal relationship between ßAPP processing and Tau pathology in vivo and without Tau transgene overexpression, (2) support the amyloidogenic cascade and (3) propose a 4-step hypothesis of prodromal AD progression.
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Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/patologia , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/metabolismo , Animais , Progressão da Doença , Feminino , Vetores Genéticos , Humanos , Potenciação de Longa Duração , Masculino , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/metabolismo , Presenilina-1/genética , Agregação Patológica de Proteínas/metabolismo , Ratos WistarRESUMO
A microfluidic microreactor for trypsin mediated transthyretin (TTR) digestion has been developed as a step towards the elaboration of a fully integrated microdevice for the detection of a rare and disabling disease, the familial transthyretin amyloidosis (ATTR) which is related to specific TTR mutations. Therefore, an enzymatic microreactor coupled to an analytical step able to monitor the mutation of TTR on specific peptide fragments would allow an accurate monitoring of the treatment efficiency of ATTR. In this study, two types of immobilized trypsin microreactors have been investigated: a new miniaturized, microfluidic fluidized bed packed with trypsin functionalized magnetic particles (MPs), and a thiol-ene (TE) monolith-based chip. Their performances were first demonstrated with N-benzoyl-dl-arginine-4-nitroanilide hydrochloride BApNA, a low molecular weight substrate. High reaction yields (75.2%) have been reached within 0.6 min for the TE-based trypsin microreactor, while a lower yield (12.4%) was obtained for the micro-fluidized bed within a similar residence time. Transposition of the optimized conditions, developed with BApNA, to TTR digestion in the TE-based trypsin microreactor was successfully performed. We demonstrated that the TE-chip can achieve an efficient and reproducible digestion of TTR. This has been assessed by MS detection. In addition, TTR hydrolysis led to the production of a fragment of interest allowing the therapeutic follow-up of more than twenty possible ATTR mutations. High sequence coverage (90%), similar to those obtained with free trypsin, was achieved in a short time (2.4 min). Repeated experiments showed good reproducibility (RSD = 6.8%). These promising results open up the route for an innovative treatment follow-up dedicated to ATTR.
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Neuropatias Amiloides Familiares/diagnóstico , Técnicas Analíticas Microfluídicas/instrumentação , Pré-Albumina/análise , Humanos , Reprodutibilidade dos TestesRESUMO
A proteomics assay was set up to analyze food substrates for eight toxins of the CBRN (chemical, biological, radiological and nuclear) threat, namely ricin, Clostridium perfringens epsilon toxin (ETX), Staphylococcus aureus enterotoxins (SEA, SEB and SED), shigatoxins from Shigella dysenteriae and entero-hemorragic Escherichia coli strains (STX1 and STX2) and Campylobacter jejuni cytolethal distending toxin (CDT). The assay developed was based on an antibody-free sample preparation followed by bottom-up LC-MS/MS analysis operated in targeted mode. Highly specific detection and absolute quantification were obtained using isotopically labeled proteins (PSAQ standards) spiked into the food matrix. The sensitivity of the assay for the eight toxins was lower than the oral LD50 which would likely be used in a criminal contamination of food supply. This assay should be useful in monitoring biological threats. In the public-health domain, it opens the way for multiplex investigation of food-borne toxins using targeted LC-MS/MS.
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Proteômica/métodos , Toxinas Bacterianas/análise , Cromatografia Líquida , Enterotoxinas/análise , Toxina Shiga/análise , Espectrometria de Massas em TandemRESUMO
Abrin expressed by the tropical plant Abrus precatorius is highly dangerous with an estimated human lethal dose of 0.1-1 µg/kg body weight. Due to the potential misuse as a biothreat agent, abrin is in the focus of surveillance. Fast and reliable methods are therefore of great importance for early identification. Here, we have developed an innovative and rapid multiepitope immuno-mass spectrometry workflow which is capable of unambiguously differentiating abrin and its isoforms in complex matrices. Toxin-containing samples were incubated with magnetic beads coated with multiple abrin-specific antibodies, thereby concentrating and extracting all the isoforms. Using an ultrasonic bath for digestion enhancement, on-bead trypsin digestion was optimized to obtain efficient and reproducible peptide recovery in only 30 min. Improvements made to the workflow reduced total analysis time to less than 3 h. A large panel of common and isoform-specific peptides was monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode on a quadrupole-Orbitrap high resolution mass spectrometer. Additionally, absolute quantification was accomplished by isotope dilution with labeled AQUA peptides. The newly established method was demonstrated as being sensitive and reproducible with quantification limits in the low ng/mL range in various food and clinical matrices for the isoforms of abrin and also the closely related, less toxic Abrus precatorius agglutinin. This method allows for the first time the rapid detection, differentiation, and simultaneous quantification of abrin and its isoforms by mass spectrometry.
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Abrina/análise , Abrina/isolamento & purificação , Fracionamento Químico/métodos , Espectrometria de Massas em Tandem , Toxinas Biológicas/análise , Toxinas Biológicas/isolamento & purificação , Abrina/química , Abrina/metabolismo , Abrus/química , Sequência de Aminoácidos , Animais , Leite/química , Modelos Moleculares , Conformação Proteica , Proteólise , Fatores de Tempo , Toxinas Biológicas/química , Toxinas Biológicas/metabolismoRESUMO
Tau protein plays a major role in neurodegenerative disorders, appears to be a central biomarker of neuronal injury in cerebrospinal fluid (CSF), and is a promising target for Alzheimer's disease immunotherapies. To quantify tau at high sensitivity and gain insights into its naturally occurring structural variations in human CSF, we coupled absolute quantification using protein standard with the multiplex detection capability of targeted high-resolution mass spectrometry (MS) on a Quadrupole-Orbitrap instrument. Using recombinant tau we developed a step-by-step workflow optimization including an extraction protocol that avoided affinity reagents and achieved the monitoring of 22 tau peptides uniformly distributed along the tau sequence. The lower limits of quantification ranged (LLOQ) from 150 to 1500 pg/mL depending on the peptide. Applied to endogenous CSF tau, up to 19 peptides were detected. Interestingly, there were significant differences in the abundance of peptides depending on their position in the sequence, with peptides from the tau mid-domain appearing significantly more abundant than peptides from the N- and C-terminus domains. This MS-based strategy provided results complementary to those of previous ELISA or Western Blot studies of CSF tau and could be applied to tau monitoring in human CSF cohorts.
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Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Peptídeos/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Sequência de Aminoácidos , Biomarcadores/líquido cefalorraquidiano , Cromatografia Líquida , Humanos , Dados de Sequência Molecular , Peptídeos/química , Reprodutibilidade dos Testes , Proteínas tau/químicaRESUMO
Bacillus anthracis is the causative bacteria of anthrax, an acute and often fatal disease in humans. The infectious agent, the spore, represents a real bioterrorism threat and its specific identification is crucial. However, because of the high genomic relatedness within the Bacillus cereus group, it is still a real challenge to identify B. anthracis spores confidently. Mass spectrometry-based tools represent a powerful approach to the efficient discovery and identification of such protein markers. Here we undertook comparative proteomics analyses of Bacillus anthracis, cereus and thuringiensis spores to identify proteoforms unique to B. anthracis. The marker discovery pipeline developed combined peptide- and protein-centric approaches using liquid chromatography coupled to tandem mass spectrometry experiments using a high resolution/high mass accuracy LTQ-Orbitrap instrument. By combining these data with those from complementary bioinformatics approaches, we were able to highlight a dozen novel proteins consistently observed across all the investigated B. anthracis spores while being absent in B. cereus/thuringiensis spores. To further demonstrate the relevance of these markers and their strict specificity to B. anthracis, the number of strains studied was extended to 55, by including closely related strains such as B. thuringiensis 9727, and above all the B. cereus biovar anthracis CI, CA strains that possess pXO1- and pXO2-like plasmids. Under these conditions, the combination of proteomics and genomics approaches confirms the pertinence of 11 markers. Genes encoding these 11 markers are located on the chromosome, which provides additional targets complementary to the commonly used plasmid-encoded markers. Last but not least, we also report the development of a targeted liquid chromatography coupled to tandem mass spectrometry method involving the selection reaction monitoring mode for the monitoring of the 4 most suitable protein markers. Within a proof-of-concept study, we demonstrate the value of this approach for the further high throughput and specific detection of B. anthracis spores within complex samples.
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Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Proteínas de Bactérias/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Biomarcadores , Cromatografia Líquida , Misturas Complexas/metabolismo , Biologia Computacional , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Especificidade da Espécie , Esporos Bacterianos/genéticaRESUMO
We have developed and describe here for the first time a highly sensitive method for the fast and unambiguous detection of viable Escherichia coli in food matrices. The new approach is based on using label-free phages (T4), obligate parasites of bacteria, which are attractive for pathogen detection because of their inherent natural specificity and ease of use. A specific immunomagnetic separation was used to capture the progeny phages produced. Subsequently, T4 phage markers were detected by liquid chromatography coupled to targeted mass spectrometry. Combining the specificity of these three methodologies is of great interest in developing an alternative to conventional time-consuming culture-based technologies for the detection of viable bacteria for industrial applications. First, optimization experiments with phage T4 spiked in complex matrices (without a phage amplification event) were performed and demonstrated specific, sensitive, and reproducible phage capture and detection in complex matrices including Luria-Bertani broth, orange juice, and skimmed milk. The method developed was then applied to the detection of E. coli spiked in foodstuffs (with a phage amplification event). After having evaluated the impact of infection duration on assay sensitivity, we showed that our assay specifically detects viable E. coli in milk at an initial count of ≥1 colony-forming unit (cfu)/mL after an 8-h infection. This excellent detection limit makes our new approach an alternative to PCR-based assays for rapid bacterial detection.
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Escherichia coli/fisiologia , Microbiologia de Alimentos/métodos , Separação Imunomagnética , Espectrometria de Massas por Ionização por Electrospray/métodos , Bacteriófago T4/química , Bacteriófago T4/isolamento & purificação , Escherichia coli/isolamento & purificação , Escherichia coli/virologia , Limite de Detecção , Fatores de TempoRESUMO
The development of rapid methods for unambiguous identification and precise quantification of protein toxins in various matrices is essential for public health surveillance. Nowadays, analytical strategies classically rely on sensitive immunological assays, but mass spectrometry constitutes an attractive complementary approach thanks to direct measurement and protein characterization ability. We developed here an innovative multiplex immuno-LC-MS/MS method for the simultaneous and specific quantification of the three potential biological warfare agents, ricin, staphylococcal enterotoxin B, and epsilon toxin, in complex human biofluids and food matrices. At least 7 peptides were targeted for each toxin (43 peptides in total) with a quadrupole-Orbitrap high-resolution instrument for exquisite detection specificity. Quantification was performed using stable isotope-labeled toxin standards spiked early in the sample. Lower limits of quantification were determined at or close to 1 ng·mL(-1). The whole process was successfully applied to the quantitative analysis of toxins in complex samples such as milk, human urine, and plasma. Finally, we report new data on toxin stability with no evidence of toxin degradation in milk in a 48 h time frame, allowing relevant quantitative toxin analysis for samples collected in this time range.
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Enterotoxinas/análise , Ricina/análise , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Animais , Isótopos de Carbono/química , Cromatografia Líquida de Alta Pressão , Enterotoxinas/sangue , Enterotoxinas/urina , Humanos , Marcação por Isótopo , Leite/química , Isótopos de Nitrogênio/química , Peptídeos/análise , Peptídeos/normas , Ricina/sangue , Ricina/urina , Espectrometria de Massas em Tandem/normasRESUMO
The metabolome is the set of small molecular mass compounds found in biological media, and metabolomics, which refers to as the analysis of metabolome in a given biological condition, deals with the large scale detection and quantification of metabolites in biological media. It is a data driven and multidisciplinary approach combining analytical chemistry for data acquisition, and biostatistics, informatics and biochemistry for mining and interpretation of these data. Since the middle of the 2000s, high resolution mass spectrometry is widely used in metabolomics, mainly because the detection and identification of metabolites are improved compared to low resolution instruments. As the field of HRMS is quickly and permanently evolving, the aim of this work is to review its use in different aspects of metabolomics, including data acquisition, metabolite annotation, identification and quantification. At last, we would like to show that, thanks to their versatility, HRMS instruments are the most appropriate to achieve optimal metabolome coverage, at the border of other omics fields such as lipidomics and glycomics.
Assuntos
Perfilação da Expressão Gênica/métodos , Espectrometria de Massas/métodos , Redes e Vias Metabólicas/fisiologia , Metaboloma/fisiologia , Metabolômica/métodos , Microquímica/métodos , Proteoma/metabolismo , Animais , HumanosRESUMO
According to the World Health Organization, food safety is an essential public health priority. In this context, we report a relevant proof of feasibility for the indirect specific detection of bacteria in food samples using unlabeled phage amplification coupled to ESI mass spectrometry analysis and illustrated with the model phage systems T4 and SPP1. High-resolving power mass spectrometry analysis (including bottom-up and top-down protein analysis) was used for the discovery of specific markers of phage infection. Structural components of the viral particle and nonstructural proteins encoded by the phage genome were identified. Then, targeted detection of these markers was performed on a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. E. coli at 1 × 10(5), 5 × 10(5), and 1 × 10(6) CFU/mL concentrations was successfully detected after only a 2 h infection time by monitoring phage T4 structural markers in Luria-Bertani broth, orange juice, and French bean stew ("cassoulet") matrices. Reproducible detection of nonstructural markers was also demonstrated, particularly when a high titer of input phages was required to achieve successful amplification. This strategy provides a highly time-effective and sensitive assay for bacterial detection.
Assuntos
Bacillus subtilis/isolamento & purificação , Bebidas/análise , Citrus sinensis , Colífagos/genética , Escherichia coli/isolamento & purificação , Produtos da Carne/análise , Sequência de Aminoácidos , Animais , Bacillus subtilis/virologia , Bebidas/microbiologia , Escherichia coli/virologia , Análise de Alimentos , Humanos , Lisogenia , Produtos da Carne/microbiologia , Dados de Sequência Molecular , Biblioteca de Peptídeos , Espectrometria de Massas por Ionização por Electrospray , Suínos , Proteínas Virais/genéticaRESUMO
Yersinia pestis is the causative agent of bubonic and pneumonic plague, an acute and often fatal disease in humans. In addition to the risk of natural exposure to plague, there is also the threat of a bioterrorist act, leading to the deliberate spread of the bacteria in the environment or food. We report here an immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) method for the direct (i.e., without prior culture), sensitive, and specific detection of Y. pestis in such complex samples. In the first step, a bottom-up proteomics approach highlighted three relevant protein markers encoded by the Y. pestis-specific plasmids pFra (murine toxin) and pPla (plasminogen activator and pesticin). Suitable proteotypic peptides were thoroughly selected to monitor the three protein markers by targeted MS using the selected reaction monitoring (SRM) mode. Immunocapture conditions were optimized for the isolation and concentration of intact bacterial cells from complex samples. The immuno-LC-SRM assay has a limit of detection of 2 × 10(4) CFU/mL in milk or tap water, which compares well with those of state-of-the-art immunoassays. Moreover, we report the first direct detection of Y. pestis in soil, which could be extremely useful in confirming Y. pestis persistence in the ground.