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MLT-MALDI TOP MS

Microbiology

QuestionAnswer
What stands for MALDI-TOF MS? Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry
how does MALDI-TOF MS work? Laser ionizes the proteins in the bacterial cell to generate charged molecules or molecule fragments. The ionized protein will accelerate and flight to the detector with opposite charge. Larger protein will have longer flight time.
What will happen if laser shot to bacteria directly? Bacterial proteins were degraded quickly by high energy before being ionized.
How to solve the protein degradation problem? Soft ionization technique. Matrix is a saturated organic compound, easily become volatilized to form ions in vaccum. It is mixed in excess with the sample before laser, Upon drying, the sample and matrix co-crystallize.
Whats for the function of the matrix? 1.Absorb the high energy from the laser. 2.Protect the bacterial protein from direct ionization. 3.Ionize the bacterial proteins indirectly through energy transfer from excited matrix molecule.
What is the common matrix for bacterial ID? α-cyano-4-hydroxycinnamic acid (HCCA)
Whats the wavelength of the laser beam? Nitrogen laser (wavelength of 337nm)
Whats the charge of the ionized proteins and the TOF tube? protein: +ve TOF tube: -ve
Larger m/z ratio protein will have? A longer drift time
Smaller m/z ratio protein will have? A shorter drift time
Whats the x-axis of the MALDI-TOF MS graph? Mass-to-charge ratio. For bacterial ID, only the proteins with mass range of 2,000 –20,000 m/z will be captured for analysis
Whats the y-axis of the MALDI-TOF MS graph? Signal intensity
General workflow of MALDI-TOF MS? (Direct transfer method) 1. Trace amount of over night single colony. 2.Smear onto a single spot of the target plate. 3.Overlay with1 μl matrix solution, dry in room air for 5 mins. 4. Put into machine.
Workflow of MALDI-TOF MS on plate extraction method? 1. Trace amount of over night single colony. 2.Smear onto a single spot of the target plate. 3.70% Formic acid, then add matrix. 4.Put into machine.
Whats does the score value stand for? >=2.000: Highly probable species identification. >1.700-1.999: secure genus identification, probable species identification. 3.<1.700:No ID
What organisms are poorly differentiate Escherichia coli and Shigella species Streptococcus pneumoniae and Streptococcus mitis gp.
Can species-level ID for salmonella spp? No, Serotyping is necessary
Identification limited by entries in database, the library used by HA hospital do not contain? highly infectious agents, e.g. Vibrio Cholerae, Bacillus anthracis, Burkholderia pseudomallei/mallei, Yersinia pestis
Whats the difficulty when ID E.coli? 1.Cannot be differentiated from Shigella spp. 2.Need to be confirmed by Lactose fermentation and Indole production.
Whats the difficulty when ID Streptococcus pneumoniae? 1.Cannot be deiferentiate S. mitis and S.oralis. 2.Need to be confirmed by optochin test susceptibility and bile solubility test
Whats the meaning of consistency category (A-C)? A: Green, species consistency B: Yellow, genus consistency C: Red, no consistency
Created by: kencho
 

 



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