MALDI-MS-TOF

Matrix Assisted Laser Desorption ionization Mass Spectroscopy Time of Flight

1. Matrix Assisted Laser Desorption Ionization Mass Spectroscopy Time Of Flight MALDI-MS-TOF Raj Kamal Vibhuti Department of Microbiology Central University of Haryana

2. Mass Spectrometer (MS) is an extremely valuable analytical techniques in which the molecules in a test sample are converted to gaseous ions that are subsequently separated in mass spectrometer according to their mass to charge (m/z) ratio and detected. The development of two ionization techniques electrospray (ESI) and Matrix Assisted Laser Desorption/ionization (MALDI). Both enabled the accurate mass determination of high molecular mass compounds as well as low molecular mass and has revolutionized. Production of ion in the gas phase. Acceleration of the ion to a specific velocity in an electrical field. Separation of the ion in a mass analyzer. Detection of each species of a particular m/z ratio. The mass analyzer may separate ion either by use of magnetic and electric field. Alternatively the time taken for ion of different masses to travel a given distance in space is measured accurately in the time of fight (TOF) mass spectrometer.

3. Component of a mass spectrometer A high vacuum system (10-6 torr OR 1 µ torr ) :- These include turbo-molecular pumps, diffusion pumps and rotary vane pump. A simple inlet :- This comprise a sample or target plate, HPLC, GC, direct chemical ionization. An ion source (To convert molecules into gas Phase Ion) :- This can be MALDI, ESI, FAB (fast atom bombardment) etc. A mass filter / Analyzer :- This can be TOF Quadrupole in trap etc. A detector :- This can be conversion by one multiplier, microchannel plate or array detector.

4. Principle of Mass Spectrometer Any moving charged species of mass ‘m’ and velocity ‘v’ will be deflected by an applied magnetic field. The magnitude of this deflection will depend on the momentum, µ of the species which is given by equation. µ=mv Species with large momentum are deflected less than those with small momentum. If a stream of atom and small molecules of identical velocity and charge but different in mass in the gaseous phase is passed through a magnetic field , the deflection experienced by each atom or molecules depends on its mass. This deflection can therefore provide an accurate measure of mass.

5. TOF Detecto r LASER-LIGHT mv2/r = zvb= Fc (Fc= zvbSin θ) Where, m = Mass v = Velocity of ionized particle r = Radius of curvature z = charge of ion (+) b = External magnetic field Fc = Centripetal Force Fc = ma2 = mv2/r = m Ѡ 2r (v = Ѡ r) = m(2∏ns)2 = m(2∏n rpm/60 )2

6. MALDI - M atrix A ssisted L aser D esorption I onization MALDI-MS, 1 st introduced in 1988 by Hillenkamp and Karas . In MALDI analysis, the analyte is 1 st co- cryslallized with a large molar excess of a matrix compound, usually an usually an ultraviolet absorbing weak organic acids after which laser radiation of this analyte , vaporization of the matrix which carries the analyte with it. The matrix therefore plays a key role by strongly absorbing the laser energy and causing indirectly, the analyte to vaporize. The matrix also serves as a proton donor and receptor, activating to ionize the analyte in both + Ve and – Ve ionization made.

7. MALDI matrix compounds

8. TOF - T ime O f F light Time of flight analyzer which measures the time required for each ion to travel to the end of the tube called the drift tube where they are detected. The time required for each ion to arrive at the detector is measured and from this the m/z is calculated.

9. Sample mixed with matrix is dried on the target plate which is introduced into high-vacuum chamber . The camera allows viewing of the position of the laser beam which can be tracked to optimize the signal . The sample/matrix is irradiated with laser pulses . The clock is started to measure time-of-flight . Ions are accelerated by the electric field to the same kinetic energy and are separated according to mass as they fly through the flight tube . Ions strike the detector either in linear (dashed arrow ) or reflectron (full arrows) mode at different times, depending on their m/z ratio . A data system controls instrument parameters, acquires signal versus time and processes the data.

10. Mechanism of MALDI MALDI provides for the non destructive vaporization and ionization of both large and small biomolecules. The analyte is 1 st co-crystallized with a large molar excess of a matrix compound, usually a UV absorbing weak organic acids. The matrix Play a key role by strongly absorbing the laser light energy and causing, indirectly the analyte to vaporize. The matrix play also serve as a proton donor and receptor, acting to ionize the analyte in both + Ve and – Ve ionization respectively.

11. MALDI- Mass Analyzer A linear time of flight (TOF) TOF reflectron Fourier transform mass analyzer TOF analysis is based on accelerating a set of ions to a detector where all of the ions are given the same amount of energy. Because the ions have the same energy, yet a different mass, the ion reach the detector at different times. The smaller ion reach the detector 1 st because of their grater velocity while the large ion take longer time to their larger mass. The analyzer is called TOF because the mass is determined from the ion’s time of flight The arrival time at the detector is dependent upon the mass, charge and kinetic energy (KE) of the ion. Since KE is equal to ½mv² or velocity v = (2KE/m)½ ion will travel a given distance, d within a time ‘t’ where t is dependent upon their mass to charge ratio (m/z).

12. TOF reflectron :- In the reflector mode, the fragment that does not retain the charge (neutral, denoted by 0°) is not deflected in the reflector but the charged fragments (•+) are deflected according to their m/z and a spectrum of the fragment (daughter) ions is recorded, albeit of a limited m/z range for each setting of the reflector voltages.

13. Fourier Transform Mass Analyzer :- The recent development of Fourier-transform ion cyclotron resonance ( FT-ICR) mass spectrometry has great potential in analysis of a wide range of biomolecules . It is potentially the most sensitive mass spectrometric technique and has very high mass resolution; >106 is observable with most instruments . The instrument also allows tandem MS to be carried out. The ions can be generated by a variety of techniques , such as an ESI or a MALDI source . FT-ICR MS is based on the principle of ions, which while orbiting in a magnetic field, are excited by radio frequency (RF ) signals. As a result, the ions produce a detectable image current on the cell in which they are trapped.

14. Fig. - Schematic diagram of the Fourier-transform ion cyclotron resonance (FT-ICR) instrument.

15. General comparison of MALDI mass analyzers

16. Analysis of protein by MALDI-TOF-MS The utility of MALDI-TOF-MS for protein and peptide analyzes lies in its ability to provide highly accurate molecular weight. Acquiring optimum MALDI data depends upon the choice of suitable matrixes and solvents the functional and structural properties of the analytes . Matrix used in MALDI-MS-TOF analysis ἀ -cyno-4-hydroxycinnamic acid (CCA)- Peptides, Glycopeptides & Small proteins 3,5-dimethoxy-4-hydroxycinnamic acid (SA)- Peptides & Small proteins 2,5-dihydroxybenzoic acid (DHB)- Glycopeptides , Glycoproteins, Small Proteins & Oligonucleotides (< 10bp)

17. Sample Preparation Pipet 0.5 µl of sample to sample plate. Pipet 0.5 µl of matrix to the sample plate. Mix the sample & matrix by drawing in the out of the pipette Allow to air dry For peptides, small proteins and most compound a saturated solution of CCA in 50:50 H2O:Acetonitrile (ACN) with 0.1% TFA ( Trifluroacetic acid) is used. For glycopeptides /proteins and small compound a saturated solution of DHB in 50:50 H2O:ACN with 0.1% TFA ( Trifluroacetic acid) is used . For prtein and large molecules a surated solution of SA in 50:50 H2O:ACN with 0.1% TFA ( Trifluroacetic acid) is used.

18. APPLICATIONS Diagnostic- MALDI a promising method for the diagnostic screening of biological fluids (serum, cerebral spinal fluid, urine ). Quantitative Aspects of Matrix-assisted Laser Desorption/Ionization- Characterizing Peptides and Reactions Directly from the Solid Phase- Several reports have shown that MS can be very useful in characterizing compounds covalently bound to solid polymeric supports subsequent to their chemical cleavage from the resin.

19. THANKS