Upload
adnya-desai
View
941
Download
7
Embed Size (px)
Citation preview
MALDI-TOFBy Miss Adnya A. DesaiDept. of Microbiology
MATRIX ASSISTED LASER DESORPTION IONIZATION-TIME OF FLIGHT
• Maldi is soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules such as DNA, proteins, peptides & sugar or polymers such as dendrimers and macromolecules
• It is three steps method. I. The sample is mixed with a suitable matrix &
applied to a metal plate.II. A pulsed laser irradiate a sample triggering
desorption of matrix material.III. Ionization of analyte molecules
HISTORY OF MS• MALDI- Term was coined in 1985 by Franz
Hillenkamp, Michael Karas• They found that amino acid alanine could be
ionized easily if it was mixed with amino acid tryptophan & irradiated with pulsed 266nm laser.
• Here, tryptophan absorbed the laser energy & helped to ionize the non absorbing alanine.
• To absorbs biomolecules more clearly Kochi Tanaka & his co-workers used ultra fine metal plus liquid matrix method that combined 30nm cobalt particles in glycerol with 337nm N2
laser for ionization. • Using this laser matrix combination Tanaka was
able to ionize biomolecule as large as 34,472 Da protein Carboxypeptidase-A. For this invention he received one quarter of the 2002 Noble Prize in Chemistry.
MASS ANALYZER
Inlet Detector DataSystem
MassAnalyzer
High Vacuum System
IonSource
Time of flight (TOF)QuadraupoleIon TrapMagnetic SectorFTMS
Turbo molecular pumps
HPLCFlow injectionSample plate
Microchannel PlateElectron MultiplierHybrid with photomultiplier
PCSun SPARK StationDEC Station
Essential Features of all MS
• Production of ions in the gas phase• Acceleration of the ions to a specific velocity
in an electric field• Separation of ions in a mass analyzer• Detection of each species of particular m/z
ratio
MALDI TOF
Camera
Laser
Sample plate
Pumping Pumping
Timed ion selector Reflector
Linear detector
Extractiongrids
Reflectordetector
Matrix
• Matrix consists of crystallized molecule of which the most 3 commonly used are 3,5dimthoxy-4-hydroxy cinnamic acid (sinapinic acid), alpha-cyano-4 hydroxycinnamic acid (CHCA/alpha-cyano/alpha-matrix), 2,5-dihydroxybenzoic acid (DHB).
• A solution of one of these molecules is made, often in a mixture of highly purified water & an organic solvent ACN i. e. acetonitril.
• Trifluoroacetic acid may also be added .• A good example of matrix solution would be 20ug/ml
sinapinic acid in ACN:water:TFA (50:50:0.1).
Structure of Matrix
LaserNumerous gas and solid state lasers have been developed for
use in MALDI.Most MALDI devices use a pulsed UV laser
N2 source at 337 nm
Neodymium-yttrium aluminum garnet (Nd:YAG)emits at 355 nm and gives a longer pulse time
IR lasers are also usedThe most common IR laser is the erbium doped-yttrium aluminum
garnet (Er:YAG)Emits at 2.94 micrometerit is “softer” than the UV, which is useful for certain biomoleculesmatrices available for IR absorption are limited
Ionization Mechanism • The mechanism remains
uncertain• It may involve absorption of light
by the matrix • Transfer of this energy to the
analyte – which then ionizes into the
gas phase as a result of the relatively large amount of energy absorbed.
– To accelerate the resulting ions into a flight-tube in the mass spectrometer they are subjected to a high electrical field [2].
Time Mass Detectors• The typical detector used with
MALDI is the time of flight mass detector (TO-FMS)
• TOF is a method where the ions are accelerated by an electric field, resulting in ions of the same strength to have the same kinetic energy .
• Time it takes for each ion to traverse the flight tube and arrive at the detector is based on its mass-to-charge ratio; therefore the heavier ions have shorter arrival times compared to lighter ions
• Ions are formed in pulses.
• The drift region is field free.
• Measures the time for ions to reach the detector.
• Small ions reach the detector before large ones.
detector
Drift region (flight tube)Source
Time-of-flight (TOF) Mass Analyzer
MALDI Advantages
see reference 8
• Gentle Ionization technique• High molecular weight analyte can be ionized• Molecule need not be volatile• Sub-picomole sensitivity easy to obtain• Wide array of matrices
TOF Advantages
• All ions detected at once• High mass accuracy and resolving power
possible• Reasonable performance for cost– <5 ppm mass accuracy and >20,000 resolving
power commercially available• High mass, low charge ions not a problem– Theoretically unlimited mass range
Reference 8
APPLICATIONS
Chemistry
Pharmaceutical analysis
Clinical analysis
Taxonomy
MICROBIOLOGY
APPLICATIONS
Proteomics Pharmaceutical Analysis• To identify, verify, and
quantitate: metabolites, recombinant proteins, proteins isolated from natural source, peptides & their amino acid sequences
• Bioavailability studies• Drug metabolism
studies, pharmacokinetics
• Characterization of potential drugs
• Drug degradation product analysis
• Screening of drug candidates
• Identifying drug targets
APPLICATIONS
Microbiology• It is used for the
identification of microorganisms.
• Species diagnosis by this procedure is much faster, more accurate & cheaper than other procedures based on biochemical tests.
Forensic analysis Environmental analysis
• Pesticides on foods• Soil and groundwater
contamination
Questions
• Who coined the term MALDI?• Name the different types of lasers are used in
maldi-tof?