MALDI-TOF Flashcards
What does the test do?
Investigates the biochemical composition of the test organism and compares test organism to reference strains and each other
What does maldi-tof stand for ?
Matrix assisted laser desorption/ ionisation- time of flight
What is good about it?
Ionisation- analysis of biomolecules intact
Broad mass range- multiple uses
Fast data acquisition
Easy to use and maintain
High sensitivity superior mass resolution and accuracy
What are the underlying principles?
It is a type of mass spec- it measures. Molecular mass of the sample
0.01% accuracy even for large biomolecules
Structural info- tandem mass spectrometers (multiple analysers)
What are the uses of mass spec?
Biotech - proteins, pep, oligoneucleotides
Pharm - drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism
Clinic - screening, Hb analysis, drug testing
Environmental - water quality, food contamin
Geological - oil composition
What is Edman degradation?
Determines the sequence of a.a. In a protein - to identify it
PITC- reacts with amino acid residues and the amino terminus under basic conditions to form PITC-protein
Acid then cleaves off the first ATZ- amino acid, converted to PTH amino acid - transferred to reverse phase HPLC C18 for detection
The chromatogram provides standard retention times of amino acids for comparison with each Edman degradation cycle chromatogram
Can understand standard composition of e.coli, work out protein sequence by weight of a.a.
Stages of maldi tof
Bacteria- Fresh agar plate to stainless steel plate (mixed in buffer/matrix- 1+ charge)
Bombarded with laser - ionises the fragments, travel along the flight tube
The variable grid and the ground grid focus the fragments into 1beam
Fragments are translated to mass:charge values (small charged molecules are faster than large) - computerised spectrum is produced
Time of flight mass analyser
What is the mass to charge ratio proportional to?
The square of its drift time
What does the tof reflector do?
The gridded ion mirror subjects ions to a uniform repulsive electric field to reflect them
The reflector compensates for the initial energy spread of ions of the same mass coming from the ion source and improves resolution
What is the problem with maldi tof?
Ions crash on to the outside of the tube they are not detected
Decomposition in time of flight tube - less obtained at the end
What is an example of a selective gate?
Bradbury Neilson gate - velocity selector - allows only selected precursor ions and their fragments to pass through to the reflector (timed ion selector)
Gates allow detection of what you want
What are the stages of sample prep?
Grown in broth culture until that reach stationary phase
Centrifuged
Pellet is re suspended in HPLC grade water
Centrifuged and re suspended in grade water
Placed on maldi with matrix solution - air dried
How is data analysed?
The generated data is compared to known organism profiles
Samples subjected to UPGMA cluster analysis - produces dendograms , show how closely related organisms are or how different
Compared by mass
When is maldi used?
Clinical applications
Environmental application on trial- identify bacteria that can clear oil spills + characterisation of legionella
