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?

REFERENCES

• www.google.com• www.slideshare.com