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Proteomic: Introduction to Mass Spectrometry proteomic- study and…

- - - - a. Time of flight (TOF)
        
        mobilised ions travel through flight tube and deflected by the reflectron
        
        (speed) is directly proportional to mass (m/z) of the analyte > smaller ions detected first
        
        increases the path length, therefore increases the resolution
      - b. Quadrupole
        
        2 different charges applied to 2 pairs of metal rods
        
        as ions travel down the quadrupole between the rods, voltages on 4 rods are scanned
        
        for a certain voltage or charge ratio, only a narrow range of m/z will pass
        
        vary in transmission modes
      - c. Ion Trap
        
        Traps ions in a 2D or 3D field
        
        Both MS and MS/MS analyses can occur.
        
        Ions are sequentially ejected to the detector
        
        high sensitivity, good range
        
        low resolution, low accuracy
        
        i. 2D ion trap (linear ion trap)
        
        Can only trap a limited number of ions (~30,000) at one time
        
        Implications for proteomics: Low resolution, very good peptide MS/MS
        
        ii. all m/z ions are trapped and detected- better sensitivity
        
        Very efficient MS/MS, especially for peptides.
        
        Can only trap a limited number of ions (~1000) at one time.
        
        Wolfgang Paul and Hans Dehmelt: Nobel Prize in Physics (1989)
      - d. Orbitrap
        
        ions are trapped between a central & an outer electrode
        
        ions oscillate to produce a current that is transformed to a m/z
        
        high sensitivity, good range
        
        very high resolution & accuracy
    - - Method
        
        a. Matrix-assisted laser desorption ionization (MALDI)
        
        heating crystals of analyte on sample plate using laser pulses to generate ions
        
        suits for determining the molecular weight of proteins (parts per million accuracy)
        
        Sample preparation before MS BY Enzymatic in-gel protein digestion
        
        mix sample with matrix and allowed to crystallize on a plate & co-crystallize with matrix molecules
        Eg of matrix molecules:
        
        α-cyano 4 hydroxycinnamic acid (CHCA)
        
        dihydroxy benzoic acid (DHB)
        
        sinapic acid
        
        3-hydroxy picolinic acid (3-HPA)
        
        PROTOCOL
        
        UV absorbing matrix is energized by the laser to generate ions
        
        Matrix molecules desorbed from the surface carry analyte molecules into the gas phase
        
        Proton transfer from matrix (acid) to sample analyte in the gas phase
        
        b. Electrospray ionization (ESI)
        
        ions created by applying a potential/ force to sample through a narrow spray needle resulting in a fine spray of ions
        
        Used to measure molecular weight, but is most powerful when used in tandem MS/MS.
        
        PROTOCOL
        
        Sample dissolved in volatile solvent of water & organic solvent mixture
        
        Sample is introduced into highly charged needle
        
        High voltage forces sample to become charged, causing liquid to break up into fine spray of small charged droplets from needle
        
        Solvent evaporation occurs leaving only smaller charged droplets
        
        Coulombic fission leads to the production of smaller droplets until individual ions are formed
        
        Generated ions are then transferred into the mass analyzer of the mass spectrometer, where they are separated based on their mass-to-charge ratio (m/z)
        
        Separated ions are detected by the mass spectrometer, and the resulting mass spectrum provides information about the mass and abundance of the ions
        
        Data analysis of acquired mass spectrum using appropriate software to interpret the results, identify the molecular ions, and determine the mass of the analyte