Mass spectrometry and MALDI TOF

The most accurate analytical technique for the measurement of the mass as well as the identification of the bio molecules

1. MASS SPECTROMETRY & MALDI-TOF By Mahek Sharan Mtech Biotechnology India

2. Contents Introduction Basic steps Basic components Types of vacuum pumps Types of ion sources Types of mass analyser MALDI TOF Matrix Laser Principle of mechanism TOF Detector Applications

3. MASS SPECTROMETRY The analytical technique by which the biological molecules are firstly converted into gaseous ions and then are separated by the mass to charge (m/z) ratio and detected This technique could accurately measure the mass of the low as well as high molecular compound by 2 techniques ESI MALDI-TOF

4. The basic steps involved Production of ions in gaseous phase Acceleration of ions in the applied electric field at specific velocity Separation of the ion in the mass analyser Detection by m/z ratio by the detectors

5. Basic component of mass spectrometry High vacuum system

6. Components High vacuum system 3 types of pumps used Turbomolecular pump Diffusion pump Rotatory vane pump Sample inlet Many types of sample or target plate are obtained by HPLC Gas Chromatography Capillary Electrophoresis Solid probe Direct chemical Ion source Electron impact ionisation Chemical ionisation Electrospray Ionisation Fast atom bombardment MALDI Mass analyser TOF Quadrupole Ion cyclotron Ion trap Magnetic sector Detector Conversion diode Electron multiplier Microchannel plate Array detector

7. Types of Vacuum pump Turbomolecular pump Uses the principle of momentum of air particles can be given and changed when it is collided by the solid surface There are rotots hitting gas molecules and making them leave from exhaust by the help of Backing pump and hevlovk pump for lowe pressure generation Diffusion pump Uses high speed jet of vapours to direct molecules out of the chamber Oil used are silicone oil or polyphenyl oil High level vacuum created

8. Rotary vane pump Uses vane fixed on rotors to make gaseous molecules leave the chamber It traps the air and later move it out by the rotation in the cavity This displacement pump is less suitable for high viscous liquids

9. Types of Ion sources Electron Impact Ionisation (EI) The metal plate / filamnet is heated in a vacuum system resulting in the positive charge of the sample in gaseous phase by the electrons released in a form of beam colliding to it Fragmentation of the ions occur into radical and ions, called Molecular ions and radicals are of no use only true ions, called Product ions ionise the samples Chemical Ionisation (CI) The ion source is the chemical involved Methane and Ammonia which are carrying the charge and can either add or take an electron to make the sample ions charged The charging of the samples done by these two chemicals serves the role in high vacuum chambers

10. Electrospray Ionisation (ESI) It is a soft ionisation technique (Liquid secondary ionisation) Uses jet spray from a capillary nozzle to ionise the sample ions by aerosol conversion in presence of inert gases like nitrogen or in vacuum The spray liquids used are the water mixed organic acids like methanol, acetonitrile The liquid turns into vapours and undergo fission and leaving analyte ions with charge causing sample molecules to charge Fast atom bombardment (FAB) This is a soft ionisation technique (secondary ion) It utilises the inert gases like Argon, Neon to ionise the sample molecules by colliding the inert gases with fast moving electrons These fast atom beam when collided with the matrix (glycerol) mixed sample ions generate the ions with charge by fragmentation of the molecule

11. Types of Mass analyser Quadrupole The separation of the ions according to the mass and charge ratio by the 4 parallel hollow cylindrical rods The rods have a particular potential applied to it as the potential changes, the ions of the sample separated according to it The ions having a particular trajectory will only reach the detector and rest will collide to the rod Ion trap mass analyser Ions are separated according to two fields applied : Magnetic and electrical The magnetic filed surrounding the ions make the mass of the ions equal and then the potential varied could be used for the separation of molecules only on the basis of the charge and vice versa The ions are trapped inside the electrodes of two types with similar radio frequency applied

12. Magnetic Sector The analyser uses the magnetic field to separate out the ions The magnetic places in between the ion beam and detector gives the ion their own trajectories based on their m/z ratios The particular m/z ratio ions are separated out by this Plasma desorption Mass analyser The plasma (nuclei without electrons in orbital) is mixed with matrix in solid state A californium atom which undergoes fission to form Ba and Tf and the two atoms move discretely in collinear opposite direction One in opposite of sample triggers a clock for start of detector and other triggers the ions to move to TOF analyser from sample foil for separation

13. Ion source and Mass analyser: MALDI-TOF Here the ion source is MALDI (Matrix Assisted Laser Desorption and Ionisation) the mass analyser is TOF

14. Matrix used in MALDI-TOF

15. Types of Laser used

16. Principle of mechanism The  laser is fired at the matrix crystals in the dried-droplet spot. The matrix absorbs the laser energy. The hot plume produced during ablation contains many species: neutral and ionized matrix molecules, protonated and deprotonated matrix molecules, matrix clusters and nanodroplets . The matrix is then thought to transfer protons to the analyte molecules, thus charging the analyte . An ion observed after this process will consist of the initial neutral molecule [M] with ions added or removed. MALDI is capable of creating singly charged ions or multiply charged ions ([ M+nH ] n+ ) depending on the nature of the matrix, the laser intensity, and/or the voltage used. The ions are then transferred to the TOF analyser

17. TOF TOF- Time of flight mass analyser The type of a mass spectrometer most widely used with MALDI due to its large mass range . MALDI-TOF instruments are often equipped with a  reflectron that reflects ions using an electric field. This increases the ion flight path, thereby increasing time of flight between ions of different m/z and increasing resolution. MALDI has been coupled with IMS-TOF MS to identify phosphorylated and non-phosphorylated peptides . Time of flight thus separates according to the m/z ratio as the lightest ion reaches first to the detector and heaviest in the last

18. Detectors The final element of the mass spectrometer is the detector The detector records either the charge induced or the current produced when an ion passes by or hits a surface. The detector used commonly is   electron multiplier  is used, though other detectors including  Faraday cups  and  ion-to-photon detectors  are also used . The number of ions leaving the mass analyzer at a particular instant is typically quite small, considerable amplification is often necessary to get a signal .   Microchannel plate detectors  are commonly used in modern commercial

19. Tandem mass spectrometry A tandem mass spectrometer is one capable of multiple rounds of mass spectrometry, usually separated by some form of molecule fragmentation Here, one mass analyzer can isolate one peptide from many entering a mass spectrometer. A second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID). A third mass analyzer then sorts the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer over time, as in a quadrupole ion trap. There are various methods for fragmenting molecules for tandem MS :   E lectron capture dissociation (ECD),  E lectron transfer dissociation (ETD),  I nfrared multiphoton dissociation (IRMPD ), etc.

20. APPLICATIONS Biochemistry Polymer chemistry Organic chemistry Microbiology Parasitology Medicine Protein complexes detection

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