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Mass Spectrometry For the Identification of ProteinBY: Muniba Safdar | Category: Genetics | Submitted: 2010-08-11 07:00:28
Article Summary: "What is Mass Spectrometer? It is a technique used to identify proteins from a sample. Its principle depends on mass, i.e. less mass of ion more will be the deflection, similarly more mass of ion, less will be the deflection (source of deflection is magnetic field or electric field). Generally, it is used to find molecular mass/m.."
Mass spectrometry is a technique used to identify proteins from a sample. Its principle depends on mass, i.e. less mass of ion more will be the deflection, similarly more mass of ion, less will be the deflection (source of deflection is magnetic field or electric field). Generally, it is used to find molecular mass/mass to charge ratio of ions in a vacuum (our molecule will pass on without any collision with air molecule). More specifically, it is used to determine mass to charge ratio.
Components of Mass Spectrometer
Mass spectrometer has three components.
1. Ion Source: molecules are converted into ions. Vacuum is created.
2. Mass Analyzer: calculation of mass to charge ratio.
3. Detector: This detects molecule/protein/peptide present.
Firstly, peptides are inserted into mass spectrometer, and then they are inserted into ion source where inert gas is introduced to avoid any reaction then collide with some material.
Normally, we use electric field in case of peptides. Peptide becomes unstable, one of the electrons will knock out and peptide becomes charged. Mass spectrometer always deals with positive charge (+1). Chlorine will also get positive charge 35.5/+1. But in case of 35.5/+2 this statement is not true.
When we have applied strong electric field, peptides are broken into fragments in ion source. These fragments will be placed into mass analyzer. Mass of fragment will be determining one by one.
To bring intact protein to mass analyzer and detector we have used a method called MALDI, Matrix Assisted Laser Desorption/Absorption Ionization.
MALDI (Matrix Assisted Laser Desorption/Absorption Ionization)
• First of all we make a solution of α-cyano-4-hydroxy cinnamic acid.
• Then it is mixed in purified water.
• Now we add organic solvent in it e.g. ethanol, this solution has some properties; it can absorb UV light (high absorbance capacity for UV light) and it is acidic (it will encourage its ionization).
• After preparation of this solution we put or peptide, each peptide has two properties; hydrophobic (dissolve in organic solvent) and hydrophilic (dissolve in water).
• We have MALDI plate, we put our solution on it, advantage is our solvent will vaporize and become crystallized.
• Peptides are embedded in crystals. Hence, peptides are safe.
• Now peptides are ready to go into vacuum,
• Then we pass on UV light. First three materials will be absorbed by crystals in the form of heat, charge will be shift on peptide, and peptide does not absorb UV light.
• Now peptides are ionized. Because of laser UV light, sublimation of crystals occurs.
• Ions are directly converted from solid to gas.
• Peptides ions (gaseous form) transfer into mass analyzer then into detector.
Four types of Mass Analyzer
Four types of analyzers are used.
1. Triple Quadropole
2. TOF (Time of Flight)
3. Ion Trap
4. FT-ICRA (Fourier Transform Ion cyclotron Resonance Analyzes).
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