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Rapid analysis of ricin using hot acid digestion and MALDI-TOF mass spectrometry.

ABSTRACT: Ricin is a protein toxin of considerable interest in forensics. A novel strategy is reported here for rapid detection of ricin based on microwave-assisted hot acid digestion and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Ricin samples are subjected to aspartate-selective hydrolysis, and biomarker peptide products are characterized by mass spectrometry. Spectra are obtained using post source decay and searched against a protein database. Several advantages are offered by chemical hydrolysis, relative to enzymatic hydrolysis, notably speed, robustness, and the production of heavier biomarkers. Agglutinin contamination is reliably recognized, as is the disulfide bond strongly characteristic of ricin.

SUBMITTER: Chen D

PROVIDER: S-EPMC7278220 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Rapid analysis of ricin using hot acid digestion and MALDI-TOF mass spectrometry.

Chen Dapeng D Bryden Wayne A WA Fenselau Catherine C

Journal of mass spectrometry : JMS 20180815 10

Ricin is a protein toxin of considerable interest in forensics. A novel strategy is reported here for rapid detection of ricin based on microwave-assisted hot acid digestion and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Ricin samples are subjected to aspartate-selective hydrolysis, and biomarker peptide products are characterized by mass spectrometry. Spectra are obtained using post source decay and searched against a protein database. Several advantages are o ...[more]

PMID: 29974543

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Project description:Dermatophytoses represent a major health burden in animals and man. Zoophilic dermatophytes usually show a high specificity to their original animal host but a zoonotic transmission is increasingly recorded. In humans, these infections elicit highly inflammatory skin lesions requiring prolonged therapy even in the immunocompetent patient. The correct identification of the causative agent is often crucial to initiate a targeted and effective therapy. To that end, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) represents a promising tool. The objective of this study was to evaluate the reliability of species identification of zoophilic dermatophytes using MALDI-TOF MS. The investigation of isolates from veterinary clinical samples suspicious of dermatophytoses suggests a good MALDI-TOF MS based identification of the most common zoophilic dermatophyte Microsporum canis. Trichophyton (T.) spp. usually achieved scores only around the cutoff value for secure species identification because of a small number of reference spectra. Moreover, these results need to be interpreted with caution due to the close taxonomic relationship of dermatophytes being reflected in very similar spectra. In our study, the analysis of 50 clinical samples of hedgehogs revealed no correct identification using the provided databases, nor for zoophilic neither for geophilic causative agents. After DNA sequencing, adaptation of sample processing and an individual extension of the in-house database, acceptable identification scores were achieved (T. erinacei and Arthroderma spp., respectively). A score-oriented distance dendrogram revealed clustering of geophilic isolates of four different species of the genus Arthroderma and underlined the close relationship of the important zoophilic agents T. erinacei, T. verrucosum and T. benhamiae by forming a subclade within a larger cluster including different dermatophytes. Taken together, MALDI-TOF MS proofed suitable for the identification of zoophilic dermatophytes provided fresh cultures are used and the reference library was previously extended with spectra of laboratory-relevant species. Performing independent molecular methods, such as sequencing, is strongly recommended to substantiate the findings from morphologic and MALDI-TOF MS analyses, especially for uncommon causative agents.

Project description:BackgroundNeuropeptides are a diverse category of signaling molecules in the nervous system regulating a variety of processes including food intake, social behavior, circadian rhythms, learning, and memory. Both the identification and functional characterization of specific neuropeptides are ongoing fields of research. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of nervous tissues from a variety of organisms allows direct detection and identification of neuropeptides. Here, we demonstrate an analysis workflow that allows for the detection of differences in specific neuropeptides amongst a variety of neuropeptides being simultaneously measured. For sample preparation, we describe a straight-forward and rapid (minutes) method where individual adult Drosophila melanogaster brains are analyzed. Using a MATLAB-based data analysis workflow, also compatible with MALDI-TOF mass spectra obtained from other sample preparations and instrumentation, we demonstrate how changes in neuropeptides levels can be detected with this method.ResultsOver fifty isotopically resolved ion signals in the peptide mass range are reproducibly observed across experiments. MALDI-TOF MS profile spectra were used to statistically identify distinct relative differences in organ-wide endogenous levels of detected neuropeptides between biological conditions. In particular, three distinct levels of a particular neuropeptide, pigment dispersing factor, were detected by comparing groups of preprocessed spectra obtained from individual brains across three different D. melanogaster strains, each of which express different amounts of this neuropeptide. Using the same sample preparation, MALDI-TOF/TOF tandem mass spectrometry confirmed that at least 14 ion signals observed across experiments are indeed neuropeptides. Among the identified neuropeptides were three products of the neuropeptide-like precursor 1 gene previously not identified in the literature.ConclusionsUsing MALDI-TOF MS and preprocessing/statistical analysis, changes in relative levels of a particular neuropeptide in D. melanogaster tissue can be statistically detected amongst a variety of neuropeptides. While the data analysis methods should be compatible with other sample preparations, the presented sample preparation method was sufficient to identify previously unconfirmed D. melanogaster neuropeptides.

Dataset Information

Rapid analysis of ricin using hot acid digestion and MALDI-TOF mass spectrometry.

Publications

Rapid analysis of ricin using hot acid digestion and MALDI-TOF mass spectrometry.

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