MALDI preparation / MS analysis: 1.) Mix sample and matrix solutions in suitable ratio. 2.) Put a sub µl aliquot of this mixture on the target plate. 3.) Let the. Cette vidéo couvre les principes de MALDI-TOF, notamment la sélection de la matrice et comment la TOF est utilisée pour élucider les rapports masse sur. MALDI is the abbreviation for “Matrix Assisted Laser Desorption/Ionization.” The sample for MALDI is uniformly mixed in a large quantity of matrix. The matrix.
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However, in recent years matrix assisted laser desorption ionization-time of flight mass spectrometry MALDI-TOF MS has emerged as a potential tool for microbial identification and diagnosis. The process is rapid, sensitive, and economical in terms of both labor and costs involved.
The technology has been readily imbibed by microbiologists who have reported usage of MALDI-TOF MS for a number of purposes like, microbial identification and strain typing, epidemiological studies, detection of biological warfare agents, detection of water- and food-borne pathogens, detection of antibiotic resistance and detection of blood and urinary tract pathogens etc. This review provides an overview of the status and recent applications of mass spectrometry for microbial identification.
MALDI-TOF/TOF-MS – WUR
It also explores the usefulness of this exciting new technology for diagnosis of diseases caused by bacteria, viruses, and fungi. The genomic information within a microbial cell translates into more than proteins, a substantial number of which can be studied using proteomics Wasinger et al. Characterization and differentiation of microbial proteome developed and progressed with both gel-based and gel-free protein separation methods.
SDS-PAGE of whole cell proteins, coupled with computer assisted analysis was used in a few studies for identification and classification of microorganisms Vandamme et al. When performed under standardized conditions, this technique was reported to be quite reproducible.
This might be attributed to: Two-dimensional gel electrophoresis 2-DE also failed to become popular among microbiologists since it was a laborious, hands-on method, even after ready availability of precast commercially produced gels and improved gel analysis softwares Cash, The merits and demerits of other quantitative proteomic approaches have been reviewed elsewhere Tiwari and Tiwari, Analysis of cellular proteome is a method which occupies an intermediary position with respect to the phenotypic—genotypic dichotomy, since the proteins analyzed reflect gene products and metabolic functions.
The increasing use of DNA fingerprinting methods in the last two decades or so has unequivocally established their applicability and utility for identification and classification of microorganisms. However, automation in proteomics technology, in recent years, has increased its throughput and potential use for a number of microbiological purposes like, strain typing and epidemiological studies, identification of microbes inhabiting a particular ecosystem, detection of biological warfare agents, detection of water- and food-borne pathogens and detection of blood and urinary tract pathogens, detection of antibiotic resistance etc.
This review provides an overview of the status and recent applications of mass spectrometry MS for microbial identification and explores the usefulness of this technology for diagnosis of diseases caused by bacteria, viruses, and fungi.
Though MS was discovered in the early s, its scope was limited to the chemical sciences.
Malsi, the development of electron spray ionization ESI and matrix assisted laser desorption ionization MALDI in s increased the applicability of MS to large biological molecules like proteins.
The sample for analysis by MALDI MS is prepared by mixing or coating with solution of an energy-absorbent, organic compound called matrix. When the matrix crystallizes on drying, the sample entrapped within the matrix also co-crystallizes. The sample within the matrix is ionized in an automated mode with a laser beam.
Desorption and ionization with the laser beam generates singly protonated ions from analytes in the sample. Princjpe charged analytes are then detected and measured using different types of mass analyzers like quadrupole mass analyzers, ion trap analyzers, time of flight TOF analyzers etc. For microbiological applications mainly TOF mass analyzers are used. A few TOF analyzers incorporate an ion mirror at pincipe rear end of the flight tube, which serves to reflect back ions through the flight tube to a detector.
Thus, the ion mirror not only increases the length of the flight tube, it also corrects small differences in energy among ions Yates, In PMF matching, the MS spectrum of unknown microbial isolates is compared with the MS spectra of known microbial isolates contained in the database. Thus, the identity of a microorganism can be established down to the genus, and in many cases to the species and strain level Fagerquist et al.
This approach is widely used in microbial identification because it is simple and can be conveniently adopted in a microbial diagnostic laboratory, aided by the availability of many commercial libraries of organism PMFs. Microbial identification by matching the biomarker masses with the molecular masses of proteins predicted from the genome sequence is not very popular in microbiological diagnostic laboratories because it requires knowledge of complete genome sequence of an organism before a database of its predicted protein molecular masses could be created.
Another research group Carbonnelle et al. The solvents penetrate the cell wall of microorganisms and extract out the intracellular proteins. The process of sample preparation for identification of microbes by MALDI-TOF MS depends upon the source from which it was isolated, or on the chemical nature of maldii constituents of its cell wall.
Investigators have evaluated different sample preparation methods for different groups of microorganisms. Some microbes might be identified directly by MS, called direct cell profiling, while for some others whole cell lysates or crude cell extracts are prepared. In direct cell profiling, a single colony of microorganism is picked and spotted directly on to the sample plate and immediately overlaid with the matrix solution.
Gram negative bacteria like Neisseria spp. Bacteria were lysed in boiling water, followed by ethanol precipitation of proteins. Researchers have reported different methods for sample preparation for identification of mycobacteria by MALDI-TOF MS, ranging from direct bacterial profiling to treatment with formic acid, safety being a major concern for routine investigations.
Mycobacterial colonies collected in screw-cap tubes containing water and 0.
Inactivated samples were centrifuged and vortexed with glass beads to disrupt the mycobacterial cell wall, the resultant pellet was re suspended in formic acid, acetonitrile, and centrifuged again. A small specimen from the mold isolate was suspended in ethanol and zirconia-silica beads, vortexed and centrifuged. Both methods reportedly gave good results in their respective studies.
This system also allowed for up gradation by providing option of adding internal libraries of organisms. Later, Andromas introduced a different kind of database and software for routine bacteriological identification which was compatible with both Bruker and Shimadzu instruments Emonet et al.
The greatest break-through in the development of MALDI-TOF MS came with its regulatory approval for routine identification of bacteria and fungi in clinical microbiological laboratories. The list of microorganisms approved for identification is unique to the individual system Patel, They are continually increasing in size and are regularly updated by the manufacturers with discovery of new microbial species and annotations.
Both, the Bruker and the Shimadzu systems contained a large collection of representative organisms in their databases and yielded comparable results with very low false positive rates Carbonnelle et al. With a few exceptions, isolates were rarely misidentified by these devices. In a few MALDI-based identification studies, some organisms could not be identified; this failure was attributed to the organism not being included in the earlier databases rather than to the methodological error Carbonnelle et al.
A critical drawback of the proprietary softwares and databases is their limited accessibility to the private researchers due to their high costs.
Nevertheless, a few research groups have developed some open-source softwares and databases which are freely available for the scientific community. Conventionally diagnosis of bacterial infections in the body fluids is carried out on the basis of biochemical and metabolic-profiling that requires 24—48 h for identification of the inflicting bacterial species.
In the meantime, patients are administered empirical antibiotics, which are sometimes inappropriate. Clinical microbiology laboratories require rapid, reliable, and cost effective methods for identification of potential pathogens in clinical samples so that appropriate antimicrobial therapy may be initiated early.
Diagnosis of infectious diarrhea in laboratory is usually done by culture and identification of bacteria in the stool samples. This is a costly and time consuming process requiring 3—5 days for detection and identification of enteric bacterial pathogens. They found that the entire procedure for identification by MALDI-TOF MS, from smear preparation to reporting of the final result was completed within 30 min, thus shortening the turnaround time of the test by 2—3 days.
Bacterial meningitis is a neurological emergency.
Matrix-assisted laser desorption/ionization
Early diagnosis is vital for rapid initiation of appropriate antimicrobial therapy. It has also been used for rapid identification of atypical, Gram-negative environmental organisms and respiratory tract pathogens which chronically infect patients with cystic fibrosis Alby et al. Rapid identification of pathogenic microorganisms is important to ensure safety and quality of water and food products. The genus Aeromonas which is indigenous to surface waters is currently composed of 17 species, of which seven can cause severe water-borne outbreaks.
MALDI-TOF MS has also been applied successfully in food microbiology for various purposes like, identification and classification of lactic acid bacteria in fermented food Nguyen et al. Tests based on biochemical traits usually fail to identify microbes isolated from environmental samples, as the diversity of microbes in these habitats is enormous Torsvik et al.
They acquired strain-specific spectra which helped in grouping aerobic and moderately halophilic prokaryotes into phenotypic clusters belonging to distinct taxa. They suggested that, if the culture conditions were not radically different, irrespective of the age or quality of the culture, MS spectrum of a microbe reflects its taxon-specific phenotypic properties which results in guaranteed identification Munoz et al.
Subdivided within three genera, these bacterial species establish either symbiotic or saprophytic interactions with plants.
The research group even constructed a database that included the type strains of currently accepted species in the family and validated it by identifying all rhizobial strains isolated from nodules and tumors Ferreira et al.
MALDI-TOF Mass Spectrometry | Protocol (Translated to French)
Fast and reliable identification of microbes which pose threats as agents of bioterrorism is required, not only to combat biological-warfare attacks, but also to prevent natural outbreaks caused by these organisms. Conventionally, organisms which pose severe threats as agents principw bioterrorism have been malldi by phenotypic, genotypic, and immunological identification systems which are slow, cumbersome and pose significant risk to the laboratory personnel.
Further work is being priincipe out to develop safe and MS-compatible protocols for inactivation of prinipe cells and spores of highly pathogenic organisms, which can be integrated into a routine microbiological laboratory.
The spores of the Bacillus spp. These were subsequently coated with the matrix solution. After the matrix solution air-dried, the spotted samples were analyzed by MS Jeong et al. In a biological war, early and unambiguous detection of toxins from aerosols is required to initiate medical countermeasures.
Nebulizer was used to generate aerosols which were collected using a cyclone collector. Tandem MS data with information from peptide sequences was used for detecting toxins that originated from organisms of any geographical location. Under careful experimental conditions, it has also been shown useful for subtyping methicillin-resistant S. Recently, Johansson et al.
Moreover using this approach they also detected the peptides specific to an aminoglycoside modifying enzyme Kan-R. Since these enzymes show different preferentiality for the CoA maldii present in different aminoglycosides, it has not been possible to establish a universal MS-based assay for detection of aminoglycoside resistance.
Conventional classification of bacteria has been carried out on the basis of biochemical, metabolic and antigenic properties. However, currently microbial species are being identified primarily on the basis of genomic information. Most DNA fingerprinting methods, however, do not correlate phylogenetically and fail to give any information regarding the evolutionary relationship between various species.
Though 16S rDNA sequencing is adequate to assign genus or species malxi an isolate, in most cases it is not sufficient for malddi typing, e. Proteomics represents the functional aspect of genomics and can be used as a taxonomic tool. Gel-based whole cell protein profiling may be as cumbersome and time consuming as any other genomic technique. In many cases it has shown resolution and reproducibility which is better than gel-based protein or DNA fingerprinting techniques van Baar, ; Fenselau and Demirev, ; Lay, The method, however, has some lacunae: During the past few years various researchers have shown applicability of MS for bacterial identification, taxonomy and strain typing.
They also determined strain-specific differences among H. Nilssondetected strain-specific biomarkers based on the analysis of six different strains of Helicobacter pylori.
Similarly, a variety of Staphylococcus spp. MALDI-TOF MS malxi of an individual microbe is the taxon-specific property of that organism, which is independent of its geographical location, culture conditions which should not be drastically different or ma,di preparation methodology.
With this approach, not pruncipe identification of new isolates as members of existing species is possible if their type strains have been previously studied Ruelle et al. The ease and rapidity in identification of large numbers of isolates can be used to study taxonomic and inter- and intra-specific diversity Feli and Dellaglio, Viruses were traditionally detected by cell culture, which in spite of being the gold standard, often took days or even weeks before any results were available.