1. M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
MASS SPECTROMETRY
M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

2. INTRODUCTION
Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles.
It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and other chemical compounds

3. PRINCIPLE The sample is ionised to generate parent molecular ions
Further fragmentation will generate fragment ions
The process of ionisation has to be controlled to generate similar ions from all the molecules in the mixture

4. The ions are separated according to their mass-to-charge ratio in an analyzer by electromagnetic fields
The ions are detected, usually by a quantitative method
The ion signal is processed into mass spectra

7. INSTRUMENTATION
The analyser, detector and ionisation source are under high vacuum to allow unhindered movement of ions
Operation is under complete data system control

8. How does a Mass Spectrometer work?
Samples easier to manipulate if ionised
Separation in analyser according to mass-to-charge ratios (m/z)
Detection of separated ions and their relative abundance
Signals sent to data system and formatted in a m/z spectrum

9. Sample introduction
Prior to sample introduction, 2 things must be achieved:
Sample must be introduced into vacuum
Sample must be vaporized
The sample is introduced by placing it on the probe, which is then inserted through a vacuum lock into ionisation region of mass spectrometer

10. Ionization Ionization means placing a charge on a neutral molecule
Methods:
1) Electron ionization
2) Electrospray
3) Matrix-assisted laser desorption/ ionization(MALDI)

11. Electron ionization
Also known as electron bombardment / electron impact method
The sample is heated to vaporize it
The sample in the gas phase is now delivered into electron ionization region
Here a beam of electrons with energy of 70EV is made to interact with the sample
This interaction causes electron ejection in the sample molecules leading to ionization

12. Electrospray
Generates ions directly from aqueous or aqueous/organic solutions
The solution is forced through a narrow needle which is kept at a high potential (3.5 kV)
The voltage on the needle causes the spray to be charged as it is nebulized
Thus, very small droplets are created and they are charged on their surfaces

13. The electric charge density on the surface of the droplet is a function of its size- smaller the droplet, larger is the electric charge density
Thus, as the droplets decrease in size, there is repulsion between mutually charged droplets
At this point, ions begin to leave the droplet
Ions are led into mass analyzer

14. Matrix-assisted laser desorption/ionization(MALDI)
It nondestructively vaporizes & ionizes both big and small molecules
The analyte is first co-crystallized with an excess of a matrix compound
Matrix compounds are organic acids, which absorb in the UV range
After the co-crystallization, a pulse UV laser beam is focused on the surface of the crystal

15. The matrix absorbs the radiation & is vaporized
The analyte is also vaporized and carried along with the matrix
The matrix doubles up as a proton acceptor or donor & thus also ionizes the analyte
Different matrices:
2,5 dihydroxy benzoic acid-proteins,peptides & oligonucleotides
Sinapinic acid – proteins & peptides

16. Interesting, right?
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