Principles and Applications of Maldi-Tof MS

PRINCIP & APPLICATIONS OF MALDI-TOF MS MATRIX-ASSISTED LASER DESORPTION IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY (MALDI-TOF MS) The term (MALDI) was coined in 1985 by Franz Hillenkamp, Michael Karas and their colleagues. A soft ionization method used for peptide mass fingerprinting. Soft ionization:- The ionization of large molecules, such as proteins and nucleic acids, without causing significant amounts of fragmentation. Time of flight (TOF) : A mass analyzer that determines the mass : charge ratio of an ion by measuring the time taken by ions to travel down a flight tube to the detector (Karas & Hillenkamp1988). • Used as a rapid, accurate, and cost-effective method for the identification of microorganisms (bacteria, fungi, and viruses). • Matrix is ionized with a laser beam, and transfers the charge to the analytes, generating singly charged ions from analytes in the sample that are then accelerated. • Ions are separated from each other on the basis of their mass-to-charge ratio (m/z) before being detected and measured using the TOF mass analyzer. • WHAT IS MATRIX? o LMW compounds which dilute the sample to dissociate the clustered macromolecule; protect the sample, absorbs the laser energy and then transfers it to the sample to avoid direct laser irradiation of the sample which can lead to decomposition of the sample molecules. • α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxy benzoic acid (DHB), and 3,5dimethoxy-4-hydroxycinnamic acid (sinapinic acid) PRINCIPLE SAMPLE PREPARATION 3/ Pick up cells from a sample strain colony and smear it on a spot 1/Pick up a part of calibration strain colony and smear it on the calibration spot 4/ 1µL Matrix solution addition on the sample spot 2/ 1µL Matrix solution addition on the calibration spot Spot visualization after crystallisation VITEK MS-DS target slide with 48 positions 2 METHOD ORGANISM TYPE INCUBATION TIME:- • 1) Bacteria - 18 - 72 hours • 2) Yeasts - 18 - 72 hours • 3) Mycobacterium - Solid Medium • Rapid growers: 3 - 7 days • • Slow growers: 7 - 28 days • 4) Nocardia - 24 - 72 hours • 5) Moulds • Rapid growers: 2 - 8 days • 6) Slow growers: 5 - 25 days • For Calibration control ATCC E.coli Strain 8739: Columbia agar + 5% sheep blood plates or Trypticase Soy agar + 5% sheep blood plates should be used. • Multiple commercial microbial identification platforms are available such as VITEK® MS system of BioMérieux, BD Bruker MALDI Biotyper System, Andromas etc. MALDI TOF MS Applications in clinical Microbiology Bacteriology 1.Clinical bacteriology 2.Detection of antibiotic resistance 3. Bacteria stain typing & taxonomy. 4.Food & water bacteriology 5.Environmental bacteriology 6.Detection of agents of biological warfare Virology Mycology 1.Clinical virology 2.Viral genotyping 3. Antiviral resistance Parasitology 1.Clinical Mycology 2. Fungal typing 3.Antifungal resistance ADVANTAGES • Significantly decreases the turnaround time. (<10 mins) and an overall 95% accuracy at the species level. • Sample preparation is simple and the sample requirement is minimal. A single colony is sufficient in order to generate spectra of sufficient quality. • Cost effective-low consumable costs • Automated, robust, interlaboratory reproducibility • Broad applicability (all types of bacteria including anaerobes, and fungi) • Adaptable-open system, expandable by user. LIMITATIONS • Identification of new isolates is possible only if the spectral database contains peptide mass fingerprints of the type strains of specific genera/species/subspecies/strains • No susceptibility information is provided • Not useful for direct testing of clinical specimens • Some organisms require repeat analysis and additional processing (extraction) • The acceptable score cutoffs vary between studies and some closely related organisms are not differentiated • Some organisms currently cannot be reliably identified by this method like E.Coli v/s Shigella, Salmonella species, Vibrio species etc.