Mass spectrometry

Mass spectrometry is an extremely valuable
analytical technique in which the molecules
in a test sample are converted into gaseous
ions that are subsequently separated in a mass
spectrometer according to their mass-to-charge
ratio (m/z) and detected .

1. Mass spectrometry is an extremely valuable analytical technique in which the molecules in a test sample are converted into gaseous ions that are subsequently separated in a mass spectrometer according to their mass-to-charge ratio (m/z) and detected .

2. Technique can be used for ……. Qualitative and quantitative analysis Providing information about the mass of atoms and molecules Molecular structure determination (organic & inorganic) Identification and characterization of materials

3. COMPONENTS OF A MASS SPECTROMETER - Sample input system - Ionization source - Mass analyzer - Detector - Vacuum pumps - Computer based data acquisition and processing system

4. The mass spectrum is a plot of a relative abundance of the ions at each m/z ratio In most cases , the nascent molecular ions of the analyte produce fragment ions by cleavage of the bond and the resulting fragmentation pattern constitute the mass spectrum

5. PRINCIPLE AND INSTRUMENTATION 1.) IONISATION Ionisation is a process of charging a molecule . The sample molecule must be charged in order to measure them using a mass spectrometer The atom is ionised by knocking one or more electrons off to give a positive ion . The particles in the sample (atoms or molecules ) are bombarded with a stream of electrons to knock one or more electrons out of the sample particles to make positive ions .

6. PRINCIPLE AND INSTRUMENTATION 2.) ACCELERATION The ions are accelerated so that they all have the same kinetic energy Ionisation chamber at +1000v The positive ions are repelled away from the positive Ionisation chamber and pass through 3 slits with voltage in the decreasing order All the ions are accelerated into a freely focused beam Ion beam

7. PRINCIPLE AND INSTRUMENTATION 3.) DEFLECTION The ions in the deflected by a magnetic field according to their masses Different ions are deflected by the magnetic field by different amounts . Amount of deflection depends upon : 1) The mass of the ion 2) The charge on the ion

8. PRINCIPLE AND INSTRUMENTATION 4.) DETECTION The beam of ions passing through the machine is detected electrically . Ions leaves space in metal by neutralizing it , & the electrons in the wire shuffle along to fill it . The flow of electrons in the wire is detected as an electric current which can be amplified & recorded

9. MASS SPECTROMETRY FOR PROTEIN ANALYSIS

10. THE TWO MOST COMMON MEATHOD OF MASS SPECTROMRTRY FOR PROTEIN ANALYSIS

11. MALDI-TOF MS The protein sample is mixed with a chemical matrix that includes a light absorbing substance excitable by a laser . The laser pulse is used to excite the chemical matrix , creating a microplasma that transfers a energy to protein molecule substance excitable by a laser . molecule in the sample ionizing them and ejecting them into the gas phase .

12. ELECTROSPARY IONISATION MASS SPECTROMETRY Electrospray is an especially soft ionisation method , capable of generating molecular ions (without fragmentation) from biological macromolecules present in aqueous solution

13. esI-MS A solution of macromolecules is sprayed in the form of fine droplets from a glass capillary under the influence of a strong electric field The droplets pick up the positive charge as they exit the capillary Evaporation of the solvent leaves multiply charged molecules

14. Analysis and Separation of Sample Ions The mass analyzer separates the ions formed in the ionization source according to their mass-to-charge ratios using some physical property e.g. Electric or magnetic fields . In MALDI-TOF the ions produced by a shorter laser pulse travel across a flight tube , arriving at different time at the detector .

15. - Employs two or more stages of mass analyzers - Example is two quadrupoles coupled in series - First analyzer selects ion (precursor ion) and second analyzer selects the fragments of the precursor ion - Used to obtain more information about the structure of fragment ions - Fragment ions may be dissociated into lighter fragment ions or converted into heavier ions by reaction with neutral molecule MS – MS (TANDEM MS) INSTRUMENTS

16. Applications of mass spectrometry to study proteins Identifying unknown proteins Protein sequencing (peptides are sequenced by generating multiple sets of proteins Identification of chemical modification (post translational modification in proteins after synthesis) Identification of organisms ( identifying bacteria by finger printing proteins )