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

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

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

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

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

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

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

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

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

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

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

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

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

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